马燕斌^,1,**, 王霞^2,**, 李换丽¹, 王平³, 张建诚¹, 文晋¹, 王新胜¹, 宋梅芳³, 吴霞^,1,*, 杨建平^,4,*1山西农业大学(山西省农业科学院)棉花研究所, 山西运城 044000
²运城学院生命科学系, 山西运城 044000
³北京市辐射中心, 北京 100875
⁴河南农业大学农学院, 河南郑州 450002

Transformation and molecular identification of maize phytochrome A1 gene (ZmPHYA1) in cotton

MA Yan-Bin^,1,**, WANG Xia^2,**, LI Huan-Li¹, WANG Pin³, ZHANG Jian-Cheng¹, WEN Jin¹, WANG Xin-Sheng¹, SONG Mei-Fang³, WU Xia^,1,*, YANG Jian-Ping^,4,*1Institute of Cotton Research, Shanxi Agricultural University (Shanxi Academy of Agricultural Sciences), Yuncheng 044000, Shanxi, China
²Department of Life Science, Yuncheng University, Yuncheng 044000, Shanxi, China
³Beijing Radiation Center, Beijing 100875, China
⁴College of Agronomy, Henan Agriculture University, Zhengzhou 450002, Henan, China

通讯作者: *杨建平, E-mail: jpyang@henau.edu.cn; 吴霞, E-mail: wuxiab8270@126.com

^**同等贡献
收稿日期:2020-06-15接受日期:2020-11-13网络出版日期:2021-06-12

基金资助:

山西省重点研发计划子课题.201703D211007-3 国家自然科学基金项目.31201253 国家自然科学基金项目.31871709 国家转基因生物新品种培育重大专项.2016ZX08010-003 山西省农业科学院应用基础研究计划.YGJPY2007 运城学院博士科研启动项目.YQ-2017 008

First author contact: ^**Contributed equally to this work
Received:2020-06-15Accepted:2020-11-13Online:2021-06-12

Fund supported:

The Shanxi Province Key Research and Development Program.201703D211007-3 The National Natural Science Foundation of China.31201253 The National Natural Science Foundation of China.31871709 The National Major Project for Developing New GM Crops.2016ZX08010-003 The Basic Research Program of Agricultural Sciences of Shanxi Academy.YGJPY2007 The Doctoral Research Project of Yuncheng College.YQ-2017 008

作者简介 About authors
马燕斌, E-mail:myb0517@163.com












摘要
为评价玉米ZmPHYA1基因在棉花种质资源改良中的价值, 本研究利用农杆菌介导法在陆地棉(Gossypium hirsutum) R15材料中进行了玉米ZmPHYA1基因的遗传转化。经过愈伤组织诱导、抗性愈伤筛选、体细胞分化诱导后获得棉花转基因再生植株。通过田间草铵膦除草剂筛选鉴定抗性植株, 并利用PCR扩增其草铵膦抗性基因和目的基因ZmPHYA1进行分子鉴定, 发现阳性植株对草铵膦除草剂具较好抗性, 并可扩增到256 bp的草铵膦抗性基因和217 bp ZmPHYA1基因的特异条带。进一步通过免疫印迹检测表明, 3个不同转基因株系中外源ZmPHYA1基因可正常表达约170 kD大小的蛋白, 且在不同组织中该外源蛋白均可正常表达。此外, 对转基因植株的不同农艺性状分析表明, 转基因株系株高明显低于受体对照, 而铃重和纤维长度等性状无明显差异。本研究成功获得具有草铵膦抗性和外源ZmPHYA1基因的棉花新种质材料, 为进一步利用光敏色素基因创新种质资源提供了材料来源。
关键词： 陆地棉;玉米光敏色素A1;蛋白表达;草铵膦抗性

Abstract
In order to evaluate the potential value of maize phytochrome A1 gene (ZmPHYA1) in the improvement of cotton germplasm resources, we transferred it into upland cotton (Gossypium hirsutum L.) R15 via Agrobacterium tumefaciens-mediated transformation with glufosinate-resistance gene as selection marker. The regenerated cotton plants were obtained through callus induction, antibiotic resistance screening and differentiation induction. After screening the regenerated plants by the herbicide glufosinate ammonium in the field, PCR detection confirmed that both the target bands, including 256 bp of the glufosinate gene and 217 bp band of ZmPHYA1 gene, were detected in the homozygous transgenic plants. In addition, the exogenous ZmPHYA1 protein of about 170 kD was also checked by immuno-blot in three transgenic cotton lines. The results showed that the specific proteins could be detected in different tissues, including leaves, flowers and stems in the transgenic Line 9. The plant height of transgenic Line 9, Line 14, Line 41 were significantly shorter than that of the wild type, while the differences of other yield-related agronomic traits were not observed between the transgenic lines and the wild type. In this study, new cotton germplasms with glufosinate resistance and ZmPHYA1 gene were successfully obtained, which provided a material source for further utilization of phytochrome gene to innovate germplasm resources.
Keywords：Gossypium hirsutum;maize phytochrome A1;protein expression;glufosinate resistance


PDF (2469KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文
本文引用格式
马燕斌, 王霞, 李换丽, 王平, 张建诚, 文晋, 王新胜, 宋梅芳, 吴霞, 杨建平. 玉米光敏色素A1基因(ZmPHYA1)在棉花中的转化及分子鉴定[J]. 作物学报, 2021, 47(6): 1197-1202. doi:10.3724/SP.J.1006.2021.03037
MA Yan-Bin, WANG Xia, LI Huan-Li, WANG Pin, ZHANG Jian-Cheng, WEN Jin, WANG Xin-Sheng, SONG Mei-Fang, WU Xia, YANG Jian-Ping. Transformation and molecular identification of maize phytochrome A1 gene (ZmPHYA1) in cotton[J]. Acta Agronomica Sinica, 2021, 47(6): 1197-1202. doi:10.3724/SP.J.1006.2021.03037


光敏色素是一类与植物发育密切相关的光信号受体蛋白, 它能通过介导光信号调控植物种子萌发、向光性、避荫性、茎秆伸长、叶绿体运动以及开花期等生长发育过程^[1,2]。光敏色素家族成员在单子叶和双子叶植物中均有报道。在拟南芥中光敏色素基因家族有PHYA、PHYB、PHYC、PHYD和PHYE共5个成员组成^[2]。而在禾本科植物中, 光敏色素基因只存在PHYA、PHYB和PHYC 3个亚家族。其中, 二倍体水稻中含有OsPHYA、OsPHYB和OsPHYC 3个单拷贝基因^[3,4], 对OsPHYA、OsPHYB和OsPHYC基因的单缺失突变体和双缺失突变体研究证实, 该基因与胚芽鞘伸长、根的向地性反应、幼苗去黄化以及开花时间等的调控相关^[5,6]。异源六倍体小麦中光敏色素基因家族中TaPHYA、TaPHYB和TaPHYC分别有3个拷贝^[7,8]。玉米由于远古种基因组经过了四倍体化的过程, 造成基因组中ZmPHYA、ZmPHYB和ZmPHYC分别保留有2个拷贝^[9,10]; 其中, 玉米中ZmPHYA1和ZmPHYA2在不同光条件下均可以正常表达, 但RNA表达丰度存在一定差异^[11]。

光敏色素基因PHYA在调节作物农艺性状发育等方面具有一定作用。研究发现, 在双子叶植物烟草和番茄中过表达单子叶植物燕麦的PHYA基因, 均导致株高显著降低^[12,13], 其中, 在转基因烟草中茎和叶柄的微管组织中能检测到高浓度的燕麦PHYA, 表明微管组织是PHYA作用的一个可能位点^[13]。转拟南芥的PHYA或PHYB到水稻或马铃薯中可以分别促进水稻籽粒或马铃薯产量发生变化^{[15,16,17,18]}。除此以外, 光敏色素PHYA和PHYB可通过ABA与茉莉酸的协同互作对耐盐性进行负向调控^[14]。由此可见, 光敏素色与作物农艺经济性状等发育过程具有较为密切的关联性, 在作物中过表达光敏色素基因对探讨作物改良具有一定的重要意义。

为探讨光敏色素基因在棉花种质资源创制中的利用价值, 我们通过农杆菌介导法转化玉米ZmPHYA1基因到棉花中。我们的研究结果显示, 外源蛋白在棉花中成功表达, 该基因能有效降低棉花株高, 表明利用光敏色素基因改良棉花具有一定的价值。

1 材料与方法

1.1 目的基因载体构建及棉花转化

利用克隆的玉米ZmPHYA1基因, 与克隆载体pMD18-T载体链接, 测序编码序列长度为3393 bp ^[11], 双酶切后与pJIM19-Bar表达载体连接, 融合9×Myc标签蛋白, 该基因由35S启动子驱动, 转化感受态细胞, 卡纳霉素筛选获得阳性克隆后, 提取质粒并酶切, 经PCR检测目标基因。将构建好的pJIM19-Bar-ZmPHYA1-Myc双元表达载体转入GV3101农杆菌中备用。将该农杆菌菌株在含有利福平和卡纳的抗生素的LB液体培养基中28℃摇床悬浮过夜培养至OD₆₀₀值为0.5, 离心收集农杆菌后, 倒去上清液, 沉淀用30 g L^-1的葡萄糖重新悬浮, 将OD₆₀₀值调整到0.2后进行侵染。

将R15棉花幼苗的下胚轴切成小段, 用含有目标基因的农杆菌侵染茎段15 min, 共培养后诱导愈伤组织。诱导后的愈伤组织在含有草铵膦的培养基上筛选形成愈伤组织, 愈伤形成后进行分化诱导获得棉花再生植株^[19]。

1.2 田间抗性检测

再生获得的棉花植株统一编号, 嫁接成活后收获T₀代种子, 将T₀代种子种植移栽, 加代自交保纯获得T₁代种子。后续世代苗期在三至四叶期连续2次叶面喷施0.05%的草铵膦除草剂, 7 d后筛选抗性转基因植株, 自交后获得抗性植株种子。抗性植株与野生型植株种植在隔离网室, 按照田间种植要求统一管理, 连续自交至纯合, 收获后用于后续试验。

1.3 转基因棉花DNA分子检测

采用改良CTAB法提取棉花材料DNA ^[20]。16 μL PCR扩增反应体系: 8 μL的2×GC buffer、0.5 μL dNTP Mix、0.5 μL引物、0.1 μL ExTaq酶、0.5 μL模板DNA和ddH₂O补足12 μL。反应程序为: 95℃预变性4 min; 94℃变性40 s, 61℃退火40 s, 72℃延伸45 s, 循环25次; 最后72℃延伸10 min, 12℃保存。1%琼脂糖凝胶电泳检测PCR产物, 凝胶成像系统照相保存。引物见表1。

Table 1
表1
表1转基因棉花植株中用于目标基因的检测引物
Table 1PCR primers of objective genes in transgenic cotton

引物 Primers	序列 Primer sequences (5′-3′)	片段长度 Expected size (bp)
Basta-F	ACCATCGTCAACCACTACATC	256
Basta-R	GCTGCCAGAAA CCCACGTCAT
ZmPHYA1-1641F	CAGCAGAAGGATGCACCCTAGGCTG	217
ZmPHYA1-1857R	CGCTTGCAGTTCGGCGAGCCCATCA

新窗口打开|下载CSV

1.4 免疫印迹分析

取PCR检测为阳性的棉花植株的幼嫩叶片, 液氮研磨约0.1 g叶片, 加蛋白提取缓冲液: 50 mmol L^-1 Tris-HCl、150 mmol L^-1 NaCl、1 mmol L^-1 EDTA、10%甘油、2 mmol L^-1 NaVO₄、0.05% Tween-20、25 mmol L^-1甘油磷酸和去离子水配制, 提取后转入1.5 mL灭菌离心管中, 离心提取上清液后, 分光光度计检测蛋白浓度, 均一化稀释备用。蛋白电泳上样前加5×Loading buffer, 样品煮沸10 min变性, 取等量样品进行SDS-PAGE电泳; 电泳完成后, 切去浓缩胶及不规则边缘, 膜与胶、滤纸放置好后转膜1.5 h、取出膜后利用5%脱脂奶粉封闭2 h、期间轻摇; 完成后保持膜湿润放入含有Myc抗体的2%脱脂奶粉中杂交2 h, TBST清洗后加二抗杂交1.5 h, 清洗后磷酸缓冲液浸泡5 min, 重新加入含有NBT和BCIP的混合液染色, 显影后清洗晾干备用。

2 结果与分析

2.1 转ZmPHYA1基因植株的获得及鉴定

将浸染后的茎段在含有草铵膦筛选的诱导培养基上进行诱导并形成愈伤, 持续诱导分化形成块状愈伤(图1-A, B)。持续诱导形成胚性愈伤后, 部分发育可得到体细胞胚(图1-C), 挑选胚状体转接诱导分化形成再生植株(图1-D)。再生株持续生长一段时间后, 将其嫁接到预先种植的健壮棉花幼苗砧木上, 成活后移入花盆中种植(图1-E)。提取具有草铵膦抗性的候选再生株系的DNA, 扩增草铵膦抗性基因。PCR电泳结果表明, 草铵膦抗性基因目标条带大小为256 bp, 表明抗性植株中该草铵膦抗性基因成功整合到转基因植株中(图1-F)。

图1

新窗口打开|下载原图ZIP|生成PPT
图1外源ZmPHYA1 基因的棉花转化与再生植株的草铵膦抗性基因检测

A: 农杆菌侵染后的愈伤筛选及诱导; B: 愈伤诱导增殖; C: 胚型愈伤分化形成的体细胞胚胎; D: 再生的幼苗; E: 再生的嫁接植株; F: 部分再生株草铵膦抗性基因的检测, 其中1、2、4分别为株系Line 9、Line 14和Line 41, 3为非转基因的再生株, “-”为受体对照, “+”为质粒阳性扩增, M: DNA marker。
Fig. 1Transformation of exogenous ZmPHYA1 gene and the PCR detection of glufosinate-resistance genes in cotton plants

A: callus screening and induction of cotton hypocotyl after Agrobacterium infection; B: callus induction and proliferation; C: somatic embyos formed from embryonic callus; D: regeneration plants; E: survived regenerated graft plants; F: PCR detection of glufosinate-resistance gene in some regenerated plants. Among them, Lane 1, 2 and 4 denote Line 9, Line 14, and Line 41, respectively, 3 denotes the non-transgenic regenerated plant, “-” denotes WT control, and “+” denotes plasmid positive amplification. M: DNA marker.


为了进一步确定转基因株系中的ZmPHYA1目的基因(图2), 生育中期, 先利用草铵膦除草剂分别涂抹植株的幼嫩叶片进行抗性检测, 结果显示: 抗性植株的叶片颜色正常, 表现为明显的抗性(图2-A~B), 而非抗性植株叶片, 则表现为明显的干枯斑块(图2-C)。

图2

新窗口打开|下载原图ZIP|生成PPT
图2转外源基因ZmPHYA1棉花草铵膦抗性株系的鉴定与PCR分子检测

A, B: 抗除草剂检测的不同阳性植株后代; C: 分离的非抗性植株后代; D: 部分抗性植株的ZmPHYA1 基因的PCR检测, 其中“+”为质粒阳性扩增, 1和2为Line 9株系的不同后代单株, 3和4为Line 14株系的不同后代单株, 5和6为Line 41株系的不同后代单株。“-”为受体对照; M: DNA marker。
Fig. 2Glufosinate resistant identification and PCR detection of exogenous ZmPHYA1 gene in cotton

A, B: different positive plant offspring detected for herbicide resistance; C: isolated offspring of non-resistant plants; D: PCR detection of ZmPHYA1 in some resistant plants. Among them, “+” denotes plasmid positive amplification, 1 and 2 denote single progeny plant of Line 9, 3 and 4 denote single progeny plant of Line 14, 5 and 6 denote single progeny plant of Line 41. “-” denotes WT; M: DNA marker.


对具有草铵膦抗性的再生植株进行ZmPHYA1基因的检测。研究结果表明, 共有17个转基因株系中可检测到217 bp长度的ZmPHYA1基因目标条带, 其中Line 9、Line 14和Line 41共3个株系的片段大小如图2-D所示, 表明该ZmPHYA1基因成功整合到转基因植株中。

2.2 转基因植株外源基因ZmPHYA1的蛋白表达检测

为了检测株系中外源ZmPHYA1蛋白的表达, 我们分别提取受体R15和转基因株系中Line 9、Line 14、Line 41株系幼嫩叶片总蛋白进行分析, 利用ZmPHYA1融合标签蛋白Myc的特异抗体进行免疫印迹杂交, 结果表明, 在转基因株系Line 9、Line 14和Line 41中能够检测到约170 kD左右的条带, 而对照R15和分离出的非抗植株中无目的条带(图3-A)。该结果表明, ZmPHYA1基因在Line 9、Line 14和Line 41株系中能够正常表达。我们同时在不同组织中检测了以上株系中ZmPHYA1的表达。免疫印迹结果表明, 转基因Line 9株系的叶、花和茎中, ZmPHYA1均能正常表达(图3-B)。

图3

新窗口打开|下载原图ZIP|生成PPT
图3不同棉花转基因株系中外源ZmPHYA1蛋白表达的免疫印迹检测

M: 蛋白marker; WT: 转基因棉花受体R15; Isolated non-transgenic line: 转基因株系分离的非抗性株; #9、#14、#41分别为转外源ZmphyA1基因的不同株系Line 9、Line 14和Line 41。“+”为检测中利用转玉米中同源基因ZmPHYA2 (3393 bp)的阳性棉花植株。
Fig. 3Immuno-blot detection of exogenous ZmPHYA1 protein expression in transgenic cotton lines

M: protein markers; WT: transgenic cotton receptor R15; non-transgenic line: non-resistant plants isolated from transgenic lines. Lane #9, #14, #41 represent Line 9, Line 14 and Line 41 of transformed exogenous ZmPHYA1 gene plants. “+” is positive cotton plant using the homologous gene ZmPHYA2 (3393 bp) in the transformed maize.

2.3 转基因植株不同农艺性状的分析

为了分析ZmPHYA1对棉花农艺性状的影响, 我们考察了转基因植株的株高、单铃籽棉重、衣分和纤维长度(图4)。统计分析表明, 对照的株高测定平均值为112 cm, 转基因株系Line 9、Line 14和Line 41的株高平均值依次为105、103和104 cm, 明显低于对照植株。

图4

新窗口打开|下载原图ZIP|生成PPT
图4转基因植株不同农艺性状的分析比较

A: 转基因株系的株高表型, 标尺为10 cm; B依次为WT、#9 (Line 9), #14 (Line 14), #41 (Line 41)株系纤维长度图, 标尺为1 cm; C、D、E和F分别为WT、#9 (Line 9)、#14 (Line 14)、#41 (Line 41)的株系平均株高、平均单铃籽棉重、衣分以及纤维长度。^*表示P < 0.05差异显著。
Fig. 4Comparison of different agronomic traits in transgenic plants

A: plant height phenotype of transgenic lines, Bar = 10 cm; B is the fiber length diagram of Line 9, Line 14 and Line 41, respectively, Bar = 1 cm. Histogram of C, D, E, and F represent the plant height, average weight of single boll seed cotton, lint percentage and fiber length of control (WT), Line 9, Line 14, and Line 41 lines, respectively. ^* indicates significant differences at P < 0.05.


而对照株、Line 9、Line 14和Line 41的单铃籽棉重平均值依次为5.6、5.4、5.1和5.1 g; 衣分平均值依次为31.1%、31.5%、34.4%和33.9%; 纤维长度依次为2.93、2.96、2.92和2.99 cm, 分析可知, 单铃籽棉重、衣分、纤维长度性状差异不显著。综合以上结果分析表明: 过表达ZmPHYA1基因可导致转基因株系株高明显降低, 但对单铃籽棉、衣分和纤维长影响不明显。

3 讨论

虽然棉花是全世界重要的经济作物之一, 但是近年来, 由于用工投入多和经济效益低等原因, 棉花种植面积不断减少。持续培育棉花优良品种和提升栽培技术是保证我国棉花的自给水平和稳定棉花种植面积的有效措施。草铵膦抗性基因和Epsps (aroA)等基因在转基因植株中分别表现出对草铵膦和草甘膦除草剂极好的抗性^{[21,22,23,24,25]}。创制抗草铵膦的棉花种质资源可以提高杂草防治效率, 从而达到生产简化、降低人力投入和提高杂草防治效率等目的^[21]。

在本研究中, 我们成功获得了具有草铵膦抗性的转基因棉花。通过分析其过程观测发现, 草铵膦筛选的愈伤诱导生长速度比使用卡那霉素筛选生长较慢, 相对试验周期较长, 但筛选诱导的阳性比率较优于卡那霉素筛选。另在幼苗期结合PCR检测和叶片抗性鉴定, 可较早的鉴定出阳性转化植株, 便于后续的管理。因此, 本试验在T₀种子收获后, 通过抗性鉴定, 后续种植可较快的鉴定出分离的抗性后代并自交直至纯合。因此, 通过对田间抗性鉴定和草铵膦抗性基因PCR检测二者相互结合的方式, 可快速的纯合阳性植株, 同时, 该选择过程也可对转基因材料的抗草铵膦特性保持稳定。

多年来, 对棉花抗除草剂、抗逆、抗病、高产等性状相关的优良基因的不断发掘,以及不同棉花基因组的测序完成, 极大的丰富了棉花分子遗传育种的理论依据^[8,25-26], 为持续改良棉花种质资源的有利农艺性状提供研究基础。

光敏色素是与色素蛋白相关的一类蛋白, 在植物中过量表达PHYA基因能够引起多效作用, 如矮化、色素增加和延迟叶片衰老等。在不同作物中过表达PHYA基因证实, 其能够影响植物株高、籽粒产量等性状^[13,15-17]。本研究中, 通过DNA水平和蛋白水平检测, 表明ZmPHYA1基因稳定插入棉花中, 可正常翻译成预期蛋白。选取其中1个材料进行不同组织的蛋白表达分析, 该蛋白可以在转基因植株的不同组织部位进行表达, 预示其可能在转基因株系中的不同组织均发挥一定的作用。另外, 预测蛋白大小约为140 kD, 但从图3中可以看到实际蛋白条带约170 kD,大于预期预测, 分析可能是蛋白翻译后修饰等原因导致分子量变大, 所以杂交条带大于预期蛋白。而本研究中转基因植株株高测定表明, 过表达ZmPHYA1能够引起棉花株高的降低, ZmPHYA1引起该表型相关的分子机制需要做进一步研究。

参考文献 View Option 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

[1] Schepens I, Duek P, Fankhauser C. Phytochrome-mediated light signalling in Arabidopsis
Curr Opin Plant Biol, 2004,7:564-569.
DOI:10.1016/j.pbi.2004.07.004URLPMID:15337099 [本文引用: 1]
The phytochrome photoreceptors regulate all major transitions during the life cycle of plants. The role of each member of the phytochrome family in Arabidopsis is starting to be understood, and a molecular description of phytochrome-regulated flowering time and shade avoidance is emerging. Recent publications have challenged some areas of well-accepted models concerning phytochrome signalling. Moreover, the importance of proteolysis during phytochrome signalling is becoming very apparent.

[2] Wang H Y, Deng X W. Dissecting the phytochrome A-dependent signaling network in higher plants
Trends Plant Sci, 2003,8:172-178.
DOI:10.1016/S1360-1385(03)00049-9URLPMID:12711229 [本文引用: 2]
Plants monitor their ambient light environment using a network of photoreceptors. In Arabidopsis, phytochrome A (phyA) is the primary photoreceptor responsible for perceiving and mediating various responses to far-red light. Several breakthroughs in understanding the signaling network mediating phyA-activated responses have been made in recent years. Here, we highlight several key advances: the demonstration that light regulates nuclear translocation of phyA and its associated kinase activity; the revelation of a transcriptional cascade controlling phyA-regulated gene expression; the detection of a direct interaction between phyA and a transcription factor; and the identification and characterization of many phyA-specific signaling intermediates, some of them suggesting the involvement of the ubiquitin-proteasome pathway.

[3] Basu D, Dehesh K Schneider-Poetsch H J, Harrington S E, McCouch S R, Quail P H. Rice PHYC gene: structure, expression, map position and evolution
Plant Mol Biol, 2000,44:27-42.
DOI:10.1023/a:1006488119301URLPMID:11094977 [本文引用: 1]
Although sequences representing members of the phytochrome (phy) family of photoreceptors have been reported in numerous species across the phylogenetic spectrum, relatively few phytochrome genes (PHY) have been fully characterized. Using rice, we have cloned and characterized the first PHYC gene from a monocot. Comparison of genomic and cDNA PHYC sequences shows that the rice PHYC gene contains three introns in the protein-coding region typical of most angiosperm PHY genes, in contrast to Arabidopsis PHYC, which lacks the third intron. Mapping of the transcription start site and 5'-untranslated region of the rice PHYC transcript indicates that it contains an unusually long, intronless, 5'-untranslated leader sequence of 715 bp. PHYC mRNA levels are relatively low compared to PHYA and PHYB mRNAs in rice seedlings, and are similar in dark- and light-treated seedlings, suggesting relatively low constitutive expression. Genomic mapping shows that the PHYA, PHYB, and PHYC genes are all located on chromosome 3 of rice, in synteny with these genes in linkage group C (sometimes referred to as linkage group A) of sorghum. Phylogenetic analysis indicates that rice phyC is closely related to sorghum phyC, but relatively strongly divergent from Arabidopsis phyC, the only full-length dicot phyC sequence available.

[4] Dehesh K, Tepperman J, Christensen A H, Quail P H. phyB is evolutionarily conserved and constitutively expressed in rice seedling shoots
Mol Gen Genet, 1991,225:305-313.
DOI:10.1007/BF00269863URLPMID:2005872 [本文引用: 1]
Southern blot analysis indicates that the rice genome contains single copies of genes encoding type A (phyA) and type B (phyB) phytochromes. We have isolated overlapping cDNA and genomic clones encoding the entire phyB polypeptide. This monocot sequence is more closely related to phyB from the dicot, Arabidopsis (73% amino acid sequence identity), than it is to the phyA gene in the rice genome (50% identity). These data support the proposal that phyA and phyB subfamilies diverged early in plant evolution and that subsequent divergence accompanied the evolution of monocots and dicots. Moreover, since rice and Arabidopsis phyB polypeptides are more closely related to one another (73% identity) than are monocot and dicot phyA sequences (63-65% identity), it appears that phyB has evolved more slowly than phyA. Sequence conservation between phyA and phyB is greatest in a central core region surrounding the chromophore attachment site, and least toward the amino-terminal and carboxy-terminal ends of the polypeptides, although hydropathy analysis suggests that the overall structure of the two phytochromes has been conserved. Gene-specific Northern blot analysis indicates that, whereas phyA is negatively regulated by phytochrome in rice seedling shoots in the manner typical of monocots, phyB is constitutively expressed irrespective of light treatment. In consequence, phyA and phyB transcripts are equally abundant in fully green tissue. Since Arabidopsis phyB mRNA levels are also unaffected by light, the present results suggest that this mode of regulation is evolutionarily conserved among phyB genes, perhaps reflecting differences in the functional roles of the different phytochrome subfamilies.

[5] Takano M, Inagaki N, Xie X Z, Yuzurihara N, Hihara F, Ishizuka T, Yano M, Nishimura M, Miyao A, Hirochika H, Shinomura T. Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice
Plant Cell, 2005,17:3311-3325.
URLPMID:16278346 [本文引用: 1]

[6] Takano M, Kanegae H, Shinomura T, Miyao A, Hirochika H, Furuya M. Isolation and characterization of rice phytochrome a mutants
Plant Cell, 2001,13:521-534.
DOI:10.1105/tpc.13.3.521URLPMID:11251094 [本文引用: 1]
To elucidate phytochrome A (phyA) function in rice, we screened a large population of retrotransposon (Tos17) insertional mutants by polymerase chain reaction and isolated three independent phyA mutant lines. Sequencing of the Tos17 insertion sites confirmed that the Tos17s interrupted exons of PHYA genes in these mutant lines. Moreover, the phyA polypeptides were not immunochemically detectable in these phyA mutants. The seedlings of phyA mutants grown in continuous far-red light showed essentially the same phenotype as dark-grown seedlings, indicating the insensitivity of phyA mutants to far-red light. The etiolated seedlings of phyA mutants also were insensitive to a pulse of far-red light or very low fluence red light. In contrast, phyA mutants were morphologically indistinguishable from wild type under continuous red light. Therefore, rice phyA controls photomorphogenesis in two distinct modes of photoperception--far-red light-dependent high irradiance response and very low fluence response--and such function seems to be unique and restricted to the deetiolation process. Interestingly, continuous far-red light induced the expression of CAB and RBCS genes in rice phyA seedlings, suggesting the existence of a photoreceptor(s) other than phyA that can perceive continuous far-red light in the etiolated seedlings.

[7] Ogihara Y, Shimizu H, Hasegawa K, Tsujimoto H, Sasakuma T. Chromosome assignment of four photosynthesis-related genes and their variability in wheat species
Theor Appl Genet, 1994,88:383-394.
DOI:10.1007/BF00223649URLPMID:24186023 [本文引用: 1]
Copy numbers of four photosynthesis-related genes, PhyA, Ppc, RbcS and Lhcb1 (*)1, in wheat genomes were estimated by slot-blot analysis, and these genes were assigned to the chromosome arms of common wheat by Southern hybridization of DNA from an aneuploid series of the cultivar Chinese Spring. The copy number of PhyA was estimated to be one locus per haploid genome, and this gene was assigned to chromosomes 4AL, 4BS and 4DS. The Ppc gene showed a low copy number of small multigenes, and was located on the short arm of homoeologous group 3 chromosomes and the long arm of chromosomes of homoeologous group 7. RbcS consisted of a multigene family, with approximately 100 copies in the common wheat genome, and was located on the short arm of group 2 chromosomes and the long arm of group 5 chromosomes. Lhcb1 (*)1 also consisted of a multigene family with about 50 copies in common wheat. Only a limited number of restriction fragments (approximately 15%) were used to determine the locations of members of this family on the long arm of group 1 chromosomes owing to the multiplicity of DNA bands. The variability of hybridized bands with the four genes was less in polyploids, but was more in the case of multigene families. RFLP analysis of polyploid wheats and their presumed ancestors was carried out with probes of the oat PhyA gene, the maize Ppc gene, the wheat RbcS gene and the wheat Lhcb1 (*)1 gene. The RFLP patterns of common wheat most closely resembled those of T. Dicoccum (Emmer wheat), T. urartu (A genome), Ae. speltoides (S genome) and Ae. squarrosa (D genome). Diversification of genes in the wheat complex appear to have occurred mainly at the diploid level. Based on RFLP patterns, B and S genomes were clustered into two major groups. The fragment numbers per genome were reduced in proportion to the increase of ploidy level for all four genes, suggesting that some mechanism(s) might operate to restrict, and so keep to a minimum, the gene numbers in the polyploid genomes. However, the RbcS genes, located on 2BS, were more conserved (double dosage), indicating that the above mechanism(s) does not operate equally on individual genes.

[8] 王霞, 马燕斌, 宋梅芳, 孟凡华, 李秀全, 杨丽, 吴霞, 杨克诚, 杨建平. 小麦TaPHYA基因亚家族的克隆及表达分析
作物学报, 2012,38:1354-1360.
[本文引用: 2]

Wang X, Ma Y B, Song M F, Meng F H, Li X Q, Yang L, Wu X, Yang K C, Yang J P. Isolation and expression patterns of TaPHYA gene subfamily in common wheat
Acta Agron Sin, 2012,38:1354-1360 (in Chinese with English abstract).
[本文引用: 2]

[9] Sheehan M J, Farmer P R, Brutnell T P. Structure and expression of maize phytochrome family homeologs
Genetics, 2004,167:1395-1405.
DOI:10.1534/genetics.103.026096URLPMID:15280251 [本文引用: 1]
To begin the study of phytochrome signaling in maize, we have cloned and characterized the phytochrome gene family from the inbred B73. Through DNA gel blot analysis of maize genomic DNA and BAC library screens, we show that the PhyA, PhyB, and PhyC genes are each duplicated once in the genome of maize. Each gene pair was positioned to homeologous regions of the genome using recombinant inbred mapping populations. These results strongly suggest that the duplication of the phytochrome gene family in maize arose as a consequence of an ancient tetraploidization in the maize ancestral lineage. Furthermore, sequencing of Phy genes directly from BAC clones indicates that there are six functional phytochrome genes in maize. Through Northern gel blot analysis and a semiquantitative reverse transcriptase polymerase chain reaction assay, we determined that all six phytochrome genes are transcribed in several seedling tissues. However, expression from PhyA1, PhyB1, and PhyC1 predominate in all seedling tissues examined. Dark-grown seedlings express higher levels of PhyA and PhyB than do light-grown plants but PhyC genes are expressed at similar levels under light and dark growth conditions. These results are discussed in relation to phytochrome gene regulation in model eudicots and monocots and in light of current genome sequencing efforts in maize.

[10] Gaut B S, Doebley J F. DNA sequence evidence for the segmental allotetraploid origin of maize
Proc Natl Acad Sci USA, 1997,94:6809-6814.
URLPMID:11038553 [本文引用: 1]

[11] 马燕斌, 李壮, 蔡应繁, 周朋, 肖阳, 黄玉碧, 付凤玲, 潘光堂, 杨克诚, 杨建平. 玉米2个光敏色素A基因的克隆、蛋白结构与光诱导表达模式
中国农业科学, 2010,43:1985-1993.
[本文引用: 2]

Ma Y B, Li Z, Cai Y F, Zhou P, Xiao Y, Huang Y B, Fu F L, Pan G T, Yang K C, Yang J P. Isolation and primary analysis of protein structures and expression patterns responding to different light treatments of two phytochrome A genes in maize (Zea mays L.)
Sci Agric Sin, 2010,43:1985-1993 (in Chinese with English abstract).
[本文引用: 2]

[12] Boylan M T, Quil P H. Oat phytochrome is biologically active in transgenic tomatoes
Plant Cell, 1989,8:765-773.
[本文引用: 1]

[13] Jordan E T, Hatfield P M, Hondred D, Talon M, Zeevaart J A, Vierstra R D. Phytochrome A overexpression in transgenic tobacco: correlation of dwarf phenotype with high concentrations of phytochrome in vascular tissue and attenuated gibberellin levels
Plant Physiol, 1995,107:797-805.
DOI:10.1104/pp.107.3.797URLPMID:7716243 [本文引用: 3]
Phytochromes are a family of related chromoproteins that regulate photomorphogenesis in plants. Ectopic overexpression of the phytochrome A in several plant species has pleiotropic effects, including substantial dwarfing, increased pigmentation, and delayed leaf senescence. We show here that the dwarf response is related to a reduction in active gibberellins (GAs) in tobacco (Nicotiana tabacum) overexpressing oat phytochrome A under the control of the cauliflower mosaic virus (CaMV) 35S promoter and can be suppressed by foliar applications of gibberellic acid. In transgenic seedlings, high concentrations of oat phytochrome A were detected in stem and petiole vascular tissue (consistent with the activity of the CaMV 35S promoter), implicating vascular tissue as a potential site of phytochrome A action. To examine the efficacy of this cellular site, oat phytochrome A was also expressed using Arabidopsis chlorophyll a/b-binding protein (CAB) and the Arabidopsis ubiquitin (UBQ1) promoters. Neither promoter was as effective as CaMV 35S in expressing phytochrome in vascular tissue or in inducing the dwarf phenotype. Collectively, these data indicate that the spatial distribution of ectopic phytochrome is important in eliciting the dwarf response and suggest that the phenotype is invoked by elevated levels of the far-red-absorbing form of phytochrome within vascular tissue repressing GA biosynthesis.

[14] Yang T, Lv R, Li J H, Lin H H, Xi D. Phytochrome A and B negatively regulate salt stress tolerance of nicotiana tobacum via ABA-jasmonic acid synergisticc ross-talk
Plant Cell Physiol, 2018,59:2381-2393.
DOI:10.1093/pcp/pcy164URLPMID:30124925 [本文引用: 1]
Light signaling and phytohormones play important roles in plant growth, development, and biotic and abiotic stress responses. However, the roles of phytochromes and cross-talk between these two signaling pathways in response to salt stress in tobacco plants remain underexplored. Here, we explored the defense response in phytochrome-defective mutants under salt stress. We monitored the physiological and molecular changes of these mutants under salt stress conditions. The results showed that phytochrome A (phyA), phytochrome B (phyB) and phyAphyB (phyAB) mutants exhibited improved salt stress tolerance compared with wild-type (WT) plants. The mutant plants had a lower electrolyte leakage (EL) and malondialdehyde (MDA) concentration than WT plants, and the effect was clearly synergistic in the phyAB double mutant plants. Furthermore, the data showed that the transcript levels of defense-associated genes and the activities of some antioxidant enzymes in the mutant plants were much higher than those in WT plants. Additionally, the results indicated that phytochrome signaling strongly modulates the expression of endogenous abscisic acid (ABA) and jasmonic acid (JA) of Nicotiana tobacum in response to salt stress. To illustrate further the relationship between phytochrome and phytohormone, we measured the expression of defense genes and phytochrome. The results displayed that salt stress and application of methyl jasmonate (MeJA) or ABA up-regulated the transcript levels of salt response-associated genes and inhibited the expression of NtphyA and NtphyB. Foliar application of inhibitors of ABA and JA further confirmed that JA co-operated with ABA in phytochrome-mediated salt stress tolerance.

[15] Thiele A, Herold M, Lenk I, Quail P H, Gatz C. Heterologous expression of Arabidopsis phytochrome B in transgenic potato influences photosynthetic performance and tuber development
Plant Physiol, 1999,120:73-82.
URLPMID:10318685 [本文引用: 2]

[16] Garg A K, Sawers R J H, Wang H, Kim J K, Walker J M, Brutnell T P, Parthasarathy M V, Vierstra R D, Wu R J. Light-regulated overexpression of an Arabidopsis phytochrome a gene in rice alters plant architecture and increases grain yield
Planta, 2006,223:627-636.
DOI:10.1007/s00425-005-0101-3URLPMID:16136335 [本文引用: 1]
The phytochromes are a family of red/far-red light absorbing photoreceptors that control plant developmental and metabolic processes in response to changes in the light environment. We report here the overexpression of Arabidopsis thaliana PHYTOCHROME A (PHYA) gene in a commercially important indica rice variety (Oryza sativa L. Pusa Basmati-1). The expression of the transgene was driven by the light-regulated and tissue-specific rice rbcS promoter. Several independent homozygous sixth generation (T(5)) transgenic lines were characterized and shown to accumulate relatively high levels of PHYA protein in the light. Under both far-red and red light, PHYA-overexpressing lines showed inhibition of the coleoptile extension in comparison to non-transgenic seedlings. Furthermore, compared with non-transgenic rice plants, mature transgenic plants showed significant reduction in plant height, internode length and internode diameter (including differences in cell size and number), and produced an increased number of panicles per plant. Under greenhouse conditions, rice grain yield was 6-21% higher in three PHYA-overexpressing lines than in non-transgenic plants. These results demonstrate the potential of manipulating light signal-transduction pathways to minimize the problems of lodging in basmati/aromatic rice and to enhance grain productivity.

[17] Kong S G, Lee D S, Kwak S N, Kim J K, Sohn J K, Kim I S. Characterization of sunlight-grown transgenic rice plants expressing Arabidopsis phytochrome A
Mol Breed, 2004,14:35-46.
[本文引用: 2]

[18] Iwamoto M, Kiyota S, Hanada A, Yamaguchi S, Takano M. The multiple contributions of phytochromes to the control of internode elongation in rice
Plant Physiol, 2011,157:1187-1195.
DOI:10.1104/pp.111.184861URLPMID:21911595 [本文引用: 1]
Although phyAphyBphyC phytochrome-null mutants in rice (Oryza sativa) have morphological changes and exhibit internode elongation, even as seedlings, it is unknown how phytochromes contribute to the control of internode elongation. A gene for 1-aminocyclopropane-1-carboxylate oxidase (ACO1), which is an ethylene biosynthesis gene contributing to internode elongation, was up-regulated in phyAphyBphyC seedlings. ACO1 expression was controlled mainly by phyA and phyB, and a histochemical analysis showed that ACO1 expression was localized to the basal parts of leaf sheaths of phyAphyBphyC seedlings, similar to mature wild-type plants at the heading stage, when internode elongation was greatly promoted. In addition, the transcription levels of several ethylene- or gibberellin (GA)-related genes were changed in phyAphyBphyC mutants, and measurement of the plant hormone levels indicated low ethylene production and bioactive GA levels in the phyAphyBphyC mutants. We demonstrate that ethylene induced internode elongation and ACO1 expression in phyAphyBphyC seedlings but not in the wild type and that the presence of bioactive GAs was necessary for these effects. These findings indicate that phytochromes contribute to multiple steps in the control of internode elongation, such as the expression of the GA biosynthesis gene OsGA3ox2, ACO1 expression, and the onset of internode elongation.

[19] 李燕娥, 朱祯, 陈志贤, 吴霞, 王伟, 李淑君, 朱玉, 焦改丽, 吴家和, 徐鸿林, 范小平, 孟晋红, 肖桂芳, 李向辉. 豇豆胰蛋白酶抑制剂转基因棉花的获得
棉花学报, 1998,10(5):3-5.
[本文引用: 1]

Li Y E, Zhu Z, Chen Z X, Wu X, Wang W, Li S J, Zhu Y, Jiao G L, Wu J H, Xu H L, Fan X P, Meng J H, Xiao G F, Li X H. Obtaining transgenic cotton plants with cowpea trypsin inhibitor gene
Cotton Sci, 1998,10(5):3-5 (in Chinese with English abstract).
[本文引用: 1]

[20] Paterson A H, Brubaker C L, Wendel J F. A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis
Plant Mol Biol Rep, 1993,11:122-127.
[本文引用: 1]

[21] 王霞, 马燕斌, 吴霞, 沈志成, 林朝阳, 李朋波, 孙璇, 王新胜, 李燕娥, 李贵全. 转G10aroA棉花株系的获得及分子生物学鉴定
中国农业科学, 2014,47:1051-1057.
[本文引用: 2]

Wang X, Ma Y B, Wu X, Shen Z C, Lin C Y, Li P B, Sun X, Wang X S, Li Y E, Li G Q. Molecular biology identification of transgenic cotton lines expressing exogenous G10aroA gene
Sci Agric Sin, 2014,47:1051-1057 (in Chinese with English abstract).
[本文引用: 2]

[22] Christ B, Hochstrasser R, Guyer L, Francisco R, Aubry S, H?rtensteiner S, Weng J K. Non-specific activities of the major herbicide-resistance gene BAR
Nat Plants, 2017,3:937-945.
DOI:10.1038/s41477-017-0061-1URLPMID:29180815 [本文引用: 1]
Bialaphos resistance (BAR) and phosphinothricin acetyltransferase (PAT) genes, which convey resistance to the broad-spectrum herbicide phosphinothricin (also known as glufosinate) via N-acetylation, have been globally used in basic plant research and genetically engineered crops (1-4) . Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids (1) , indirect effects of BAR-containing transgenes in planta, including modified amino acid levels, have been seen but without the identification of their direct causes (5,6) . Combining metabolomics, plant genetics and biochemical approaches, we show that transgenic BAR indeed converts two plant endogenous amino acids, aminoadipate and tryptophan, to their respective N-acetylated products in several plant species. We report the crystal structures of BAR, and further delineate structural basis for its substrate selectivity and catalytic mechanism. Through structure-guided protein engineering, we generated several BAR variants that display significantly reduced non-specific activities compared with its wild-type counterpart in vivo. The transgenic expression of enzymes can result in unintended off-target metabolism arising from enzyme promiscuity. Understanding such phenomena at the mechanistic level can facilitate the design of maximally insulated systems featuring heterologously expressed enzymes.

[23] Siruguri V, Bharatraj D K, Vankudavath R N, Mendu V V R, Gupta V, Goodman R E. Evaluation of Bar, Barnase, and Barstar recombinant proteins expressed in genetically engineered Brassica juncea (Indian mustard) for potential risks of food allergy using bioinformatics and literature searches
Food Chem Toxicol, 2015,83:93-102.
DOI:10.1016/j.fct.2015.06.003URLPMID:26079618 [本文引用: 1]
The potential allergenicity of Bar, Barnase, and Barstar recombinant proteins expressed in genetically engineered mustard for pollination control in plant breeding was evaluated for regulatory review. To evaluate the potential allergenicity of the Bar, Barnase and Barstar proteins amino acid sequence comparisons were made to those of known and putative allergens, and search for published evidence to the sources of the genes using the AllergenOnline.org database. Initial comparisons in 2012 were performed with version 12 by methods recommended by the Codex Alimentarius Commission and the Indian Council of Medical Research, Government of India. Searches were repeated with version 15 in 2015. A literature search was performed using PubMed to identify reports of allergy associated with the sources of the three transgenes. Potential open reading frames at the DNA insertion site were evaluated for matches to allergens. No significant sequence identity matches were identified with Bar, Barnase or Barstar proteins or potential fusion peptides at the genomic-insert junctions compared to known allergens. No references were identified that associated the sources of the genes with allergy. Based on these results we conclude that the Bar, Barnase and Barstar proteins are unlikely to present any significant risk of food allergy to consumers.

[24] Carbonari C A, Latorre D O, Gomes G L G C, Velini E D, Owens D K, Pan Z Q, Dayan FD E. Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink and WideStrike cotton
Planta, 2016,243:925-933.
DOI:10.1007/s00425-015-2457-3URLPMID:26733464 [本文引用: 1]
MAIN CONCLUSION: Insertion of the gene encoding phosphinothricin acetyltransferase (PAT) has resulted in cotton plants resistant to the herbicide glufosinate. However, the lower expression and commensurate reduction in PAT activity is a key factor in the low level of injury observed in the WideStrike((R)) cotton and relatively high level of resistance observed in LibertyLink((R)) cotton. LibertyLink((R)) cotton cultivars are engineered for glufosinate resistance by overexpressing the bar gene that encodes phosphinothricin acetyltransferase (PAT), whereas the insect-resistant WideStrike((R)) cultivars were obtained using the similar pat gene as a selectable marker. The latter cultivars carry some level of resistance to glufosinate which enticed certain farmers to select this herbicide for weed control with WideStrike((R)) cotton. The potency of glufosinate on conventional FM 993, insect-resistant FM 975WS, and glufosinate-resistant IMACD 6001LL cotton cultivars was evaluated and contrasted to the relative levels of PAT expression and activity. Conventional cotton was sensitive to glufosinate. The single copy of the pat gene present in the insect-resistant cultivar resulted in very low RNA expression of the gene and undetectable PAT activity in in vitro assays. Nonetheless, the presence of this gene provided a good level of resistance to glufosinate in terms of visual injury and effect on photosynthetic electron transport. The injury is proportional to the amount of ammonia accumulation. The strong promoter associated with bar expression in the glufosinate-resistant cultivar led to high RNA expression levels and PAT activity which protected this cultivar from glufosinate injury. While the insect-resistant cultivar demonstrated a good level of resistance to glufosinate, its safety margin is lower than that of the glufosinate-resistant cultivar. Therefore, farmers should be extremely careful in using glufosinate on cultivars not expressly designed and commercialized as resistant to this herbicide.

[25] Hérouet C, Esdaile D G, Mallyon B A, Debruyne E, Schulz A, Currier T, Hendrickx K, Klis R J V D Rouan D. Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate- ammonium herbicide in transgenic plants
Regul Toxicol Pharm, 2005,41:134-149.
[本文引用: 2]

[26] Li F, Fan G, Lu C, Xiao G, Zou C, Kohel R J, Ma Z, Shang H, Ma X, Wu J, Liang X, Huang G, Percy R G, Liu K, Yang W, Chen W, Du X, Shi C, Yuan Y, Ye W, Liu X, Zhang X, Liu W, Wei H, Wei S, Huang G, Zhang X, Zhu S, Zhang H, Sun F, Wang X, Liang J, Wang J, He Q, Huang L, Wang J, Cui J, Song G, Wang K, Xu X, Yu J Z, Zhu Y, Yu S. Genome sequence of cultivated upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution
Nat Biotechnol, 2015,33:524-530.
DOI:10.1038/nbt.3208URLPMID:25893780 [本文引用: 1]
Gossypium hirsutum has proven difficult to sequence owing to its complex allotetraploid (AtDt) genome. Here we produce a draft genome using 181-fold paired-end sequences assisted by fivefold BAC-to-BAC sequences and a high-resolution genetic map. In our assembly 88.5% of the 2,173-Mb scaffolds, which cover 89.6% approximately 96.7% of the AtDt genome, are anchored and oriented to 26 pseudochromosomes. Comparison of this G. hirsutum AtDt genome with the already sequenced diploid Gossypium arboreum (AA) and Gossypium raimondii (DD) genomes revealed conserved gene order. Repeated sequences account for 67.2% of the AtDt genome, and transposable elements (TEs) originating from Dt seem more active than from At. Reduction in the AtDt genome size occurred after allopolyploidization. The A or At genome may have undergone positive selection for fiber traits. Concerted evolution of different regulatory mechanisms for Cellulose synthase (CesA) and 1-Aminocyclopropane-1-carboxylic acid oxidase1 and 3 (ACO1,3) may be important for enhanced fiber production in G. hirsutum.