,2Design and exploration of genetic comprehensive experiments based on Ds insertion mutants
Nan Li1, Yajuan Li1, Haibin Guo1, Xiangqian Zhang,2通讯作者: 张向前，博士，副教授，研究方向：植物分子育种。E-mail:aacrav@163.com
编委: 陈德富
收稿日期:2019-09-26修回日期:2019-11-1网络出版日期:2019-12-20
| 基金资助: |
Editorial board:
Received:2019-09-26Revised:2019-11-1Online:2019-12-20
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作者简介 About authors
李楠,硕士,实验师,研究方向：遗传学实验教学与管理。E-mail:523155900@qq.com。
摘要
学生创新能力的培养已成为中国高等教育的重要导向,加强综合设计性实验的应用是提高大学生创新能力的有效途径。文章以水稻Ds插入突变体为实验材料,以突变体表型观测作为实验切入点,以遗传分析和Ds转座检测为实验学习重点,重新设计了一个分子遗传学综合性实验项目。在此基础上,通过知识拓展环节引导学生利用开放实验平台学习TAIL-PCR技术,进一步进入研究性实验。通过综合性实验到研究性实验的递进式教学体系,能够使学生深入理解表型与基因的内在联系、跳跃基因的概念以及转座子在基因功能研究中的重要作用,加深学生对基因概念及遗传规律的认识和理解,提升学生理论知识与实验技能的整合运用能力。
关键词：
Abstract
The cultivation of innovative abilities has become an important guide for higher education in China. Strengthening the integrated knowledge to design experiments is an effective way to improve undergraduate students’ innovative abilities. Herein we designed a comprehensive experiment for molecular genetics by utilizing a rice Ds insertion mutant identified previously in our research project. In the comprehensive experiment, we adopt the method of scientific research as the main line of teaching and take the interesting phenotype of the rice mutant as the breakthrough point to reform and innovate genetics laboratory teaching. On the basis of this, we combined the progressive teaching method and guided the students to learn the TAIL-PCR skill and conduct an innovative experiment through expanding their knowledge. The comprehensive experiment will deepen students’ understandings of the relationship between genotypes and phenotypes, help them master the effective way of thinking and technologies for scientific research to further improve their ability of the integrated application capability of theory and practices.
Keywords：
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本文引用格式
李楠, 李亚娟, 郭海滨, 张向前. Ds插入突变体的遗传学综合性实验设计与探讨. 遗传[J], 2019, 41(12): 1148-1155 doi:10.16288/j.yczz.19-149
Nan Li.
遗传学是探究生物遗传和变异规律的理论科学,是生命科学领域的基础学科,研究内容丰富,小到基因结构、功能及表达,大到生物群落的遗传特征[1]。遗传学理论与实践关系密切,实验不仅是遗传学的重要基础,更是培养创新能力的重要环节[2]。为提升遗传学实验教学质量和促进创新人才培养,华南农业大学于2007年构建了“一般性实验—综合性实验—研究性实验”的递进式遗传学实验教学新体系,提出适合华南农业大学农科类专业的“创新型混合教学模式”[3]。2012年,华南农业大学实现了遗传学实验教学的独立设课,新编《遗传学综合性实验》教材和实验教学大纲,将遗传学实验从16学时的一般性实验调整至32学时的普通遗传学实验和48或64学时的遗传学综合设计性实验。
当前华南农业大学本科遗传学实验教学主要分为3大类综合性实验项目：细胞遗传学综合性实验、经典遗传学综合性实验及分子遗传学综合性实验。分子遗传学综合性实验从2012年应用至今已有7年,以籼粳水稻叶片为材料,利用特定SSR引物扩增籼粳水稻的DNA片段,实现对不同材料的区分。该实验主要以植物DNA提取、PCR扩增和凝胶电泳检测等分子标记相关技术的学习为主,已远不能满足创新型人才培养需要。
为提升遗传学综合设计性实验项目的质量,适应当代教学的需要,利用水稻Ds插入突变体材料,以培养学生科研思维和研究方法作为实验教学目标,以遗传分析法和PCR检测技术为学习重点,重新设计了分子遗传学综合性实验。使学生通过本实验项目的学习,能够理解表型与基因的内在联系,了解跳跃基因的概念以及转座子的重要应用价值,掌握科学研究的思维方式与研究方法,提升对理论与实践、知识与技术的整合运用能力。
1 实验材料的背景
粒重是决定水稻产量的3个主要因素(即每株有效穗数、每穗粒数和粒重)之一,是粒长、粒宽和粒厚的综合指标。而水稻粒长、粒宽及长宽比又称为水稻谷粒形状,是衡量稻米外观品质的重要指标之一,同时还影响稻米的商品品质和加工品质(糙米率、精米率和整精米率)。近年来,水稻粒形相关基因的研究取得了突破性进展。目前,至少已有93个水稻粒形相关基因被克隆,遍布水稻12条染色体[4,5,6,7]。本实验所用的突变体材料pex1是一个Ds转座引起的粒型突变体,其谷壳大小与野生型无异,而颖果显著缩小,该突变体表型为不完全显性遗传;利用转座子标签法,采用TAIL-PCR[8] (thermal asymmetric interlaced PCR)技术已获知候选基因OsPEX1位于水稻第11号染色体,该基因属于类伸展蛋白基因家族,是一个控制水稻重要农艺性状的新基因。该材料是由Ac/Ds双因子转座系统转化至粳型品种中花11号获得[9],其中用于Ds转化的质粒为pDsBar1300,用于Ac转化的质粒为pUbiTs (图1)[9]。pDsBar1300质粒的T-DNA区中携带Ds转座子,Ds的内部插入Bar 基因,能提供对Basta (商用除草剂,有效成分为膦丝菌素PPT)的抗性,在Ds的旁侧插入潮霉素磷酸转移酶基因(hygromycin phostransferase, HPT),能提供对潮霉素的抗性。将筛选得到的水稻Ds转化纯合体和Ac转化纯合体进行杂交,构建了多个杂交组合,通过Ac转座酶的反式活化使Ds发生转座[9]。
图1
新窗口打开|下载原图ZIP|生成PPT图1质粒T-DNA区和TAIL-PCR引物所在位置示意图
A：质粒pDsBar1300中T-DNA区及PCR检测引物位置示意图。HPT：潮霉素抗性基因;35S-Pro：35S启动子;Bar：Bar 基因能提供对Basta的抗性;tra4、P5和P10：Ds转座检测的3条引物。B：质粒pUbiTs中T-DNA区示意图。Ubi-Pro：Ubi启动子;Nos-Pro：Nos启动子;NPTII：NPTII基因编码新霉素磷酸转移酶,能赋予细胞抗卡那霉素的能力。LB和RB：T-DNA的左右边界。C：TAIL-PCR引物所在位置示意图。
Fig. 1Schematic diagram of T-DNA regions of the plasmids and primer placement in TAIL-PCR
2 实验设计
2.1 设计思路
利用该材料进行综合性实验设计,先将以上科学研究的实验材料与野生型杂交获得F1,并自交获得F2分离群体,以该突变体、野生型、F1杂合体、F2群体的成熟谷粒及对应水稻叶片作为课程实验材料。首先让学生通过观察与考种实验发现突变体、野生型和F1杂合体在谷壳和米粒上的异同;其次提供F2群体米粒,通过米粒宽度测量及方差分析,引导学生对该材料进行表型观察和遗传分析;再次提供学生F2群体水稻叶片,利用PCR检测技术,分析突变体表型与Ds插入之间的关系,验证表型调查结果。最后通过知识拓展环节,结合理论教学让学生深入了解转座因子在基因功能研究中的应用价值。并以pex1突变体为例,探讨如何实现该类型材料(例如T-DNA或转座子插入突变体)的基因克隆及功能分析。该实验整个教学设计基于问题引导式教学模式(problem-based learning),以问题为导向,引导学生在实验中发现问题、分析问题、提出问题、研究问题并解决问题,最后得出科学研究结论,进而培养学生科学研究的思维方式,提升学生对理论与实践、知识与技术的整合运用能力和创新实践能力。2.2 实验涉及的主要理论知识与实验技术
该实验涉及的理论知识点与实验技术主要包括孟德尔遗传定律、转座子及TAIL-PCR技术。分离定律是孟德尔遗传定律之一。在杂合子细胞中,位于一对同源染色体上的等位基因,在进行减数分裂时等位基因会随着同源染色体的分开而分离,分别进入两个配子中,独立地随配子遗传给后代,在F2代性状发生3∶1或1∶2∶1的分离[10]。转座子(transposon)是染色体上可复制和位移的一段DNA序列。转座子可以通过切割、重新整合等一系列过程从基因组的一个位置“跳跃”到另一个位置。在各种功能基因组学研究方法中,利用转座子插入诱变的方法被认为是进行大规模基因功能鉴定的有力工具[11],目前已有多例用Ac/Ds转座系统成功分离水稻基因的报道[12,13]。
TAIL-PCR技术,即交错式热不对称PCR,通过利用已知的DNA序列设计一组特异性引物,并结合随机简并引物,采用高温特异性扩增与低温随机扩增相结合的方法,获得转座子插入侧翼区特异扩增片段,相应扩增片段可直接测序分析,筛选分离基因。
2.3 实验材料与方法
2.3.1 实验材料将pex1突变体与野生型杂交,并将F1自交获得的F2群体,以突变体、野生型、F1杂合体及F2群体的成熟谷粒,F2群体谷粒对应的水稻叶片作为课程实验材料。所有材料均种植于华南农业大学农场,单株种植,常规管理。
2.3.2 实验方法
2.3.2.1 突变体的鉴定和遗传分析
一个实验班人均约30人,将学生以5人为一组,提供给每组同学野生型、突变纯合体、突变杂合体谷粒各1袋,每袋100粒。F2群体210株对应收获种子210袋,每袋100粒。考种方法为10粒为1组,3次重复,整齐摆放,配置游标卡尺与分析天平测量。利用ImageJ软件获取考种数据,并运用SPSS18.0软件进行卡方检验和t检验。
2.3.2.2 Ds转座的PCR检测
Ds转座的PCR检测参照刘芳等[9]方法。Ds因子切离的PCR检测利用引物P10、Tra4和P5同时扩增。如果Ds未发生切离,称为满载供体位点(full donor site, FDS),通过引物P10和P5可以扩增出约400 bp的特异带;如果Ds从T-DNA上切离,称为空载供体位点(empty donor site, EDS)引物Tra4和P5可以扩增出约870 bp的特异带;如果既存在EDS又存在FDS,则利用3条引物可以同时扩增出两条特异带。
具体实验步骤包括基因组DNA提取、PCR扩增、电泳分离、数据统计与分析等。每年在田间种植两亲本各20株、F120株和F2群体210株。待上课前1周取F2群体每株上部叶片置于超低温冰箱中保存备用。
水稻DNA提取：取2~4 cm长的叶片用液氮研磨至粉末,加入TPS抽提液(100 mmol/L Tris-HCl pH8.0,10 mmol/L EDTA,1 mol/L KCl)1000 μL,75℃水浴30 min;13 000r/min离心12 min,吸上清约500 μL转入1.5 ml离心管中,加入等体积遇冷的异丙醇,-20℃放置10 min,13 000 r/min离心5 min,弃上清,干燥沉淀,加200 μL灭菌水溶解,4℃冰箱冷藏备用。
PCR扩增及琼脂糖凝胶电泳检测：学生使用自己提取的基因组DNA为模板进行PCR扩增,每个模板使用3条引物(P10、Tra4和P5)同时扩增。扩增后的PCR产物用1%的琼脂糖凝胶进行电泳,在凝胶成像系统上观察电泳结果,记录样品基因型。
2.3.2.3 突变体基因克隆
利用水稻的Ac/Ds双因子转座系统,筛选Ds转座插入纯合体植株为材料,采用TAIL-PCR[8]的方法扩增Ds元件旁侧的水稻基因组序列,分析Ds插入位点旁邻序列,通过序列比对确定该基因在水稻染色体上的具体位置,并对候选基因进行了分析。TAIL-PCR反应所用引物见表1,其位置示意图如图1所示,特异扩增得到的第3轮PCR产物用来测序。Ds在水稻基因组中的插入位置运用BLAST (http://www.ncbi.nlm.nih.gov/BLAST)分析工具完成。Ds因子插入所在位点相关基因预测信息可以在水稻基因组注释数据库获得(http://rice.plantbiology.msu.edu/)。
Table 1
表1
表1实验所用引物
Table 1
| 引物 | 引物序列(5′→3′) | 实验目的 | 备注 |
|---|---|---|---|
| P10 | TCCCGTCCGATTTCGACTTTA | Ds转座检测 | |
| Tra4 | TAGCTCACTCATTAGGCACCC | Ds转座检测 | |
| P5 | AAGCTCAAGCTGCTCTAGCATTCG | Ds转座检测 | |
| Ds3′-1a | GGTTCCCGTCCGATTTCGACT | TAIL-PCR | DsR-primer |
| Ds3′-2a | CGATTACCGTATTTATCCCGTTC | TAIL-PCR | DsR-primer |
| Ds3′-3a | TCGTTTCCGTCCCGCAAGT | TAIL-PCR | DsR-primer |
| AD3 | WGTGNAGWANCANAGA | TAIL-PCR | AD primer |
| AD13 | NTSGASNTCNGAATCA | TAIL-PCR | AD primer |
新窗口打开|下载CSV
2.4 实验结果与讨论
2.4.1 突变体鉴定与遗传分析水稻pex1是1个Ds转座子插入引起的颖果明显变小,而谷粒大小没有改变的突变体(图2A)。进一步分析表明,pex1突变纯合体颖果(20.25±0.26 mm,10粒)比野生型颖果宽(27.10± 0.43 mm,10粒)减少了25.3%,而其百粒重较野生型减少了33.6%;杂合体颖果比野生型宽度减少了18.5%,百粒重减少了25.2%。
为了研究突变体表型遗传特性,我们构建了F2分离群体。在F2群体中,不同植株的颖果表型明显不同,其中60株颖果宽度类似野生型,103株颖果宽度达22.09± 0.21 mm (10粒),47株颖果宽度则与突变纯合体无异。卡方测验表明其分离比符合1∶2∶1 (χ2 = 1.68<χ20.05,2 = 5.99),说明pex1突变表型由1对不完全显性基因调控。
2.4.2 突变性状与Ds共分离分析
为了分析突变体表型与Ds之间的关系,并验证表型调查结果,对F2分离群体做了Ds转座的PCR检测(图2B)。在F2代的210个单株的分离群体中,60株野生型植株均为Ds未转座类型,47株突变体植株为Ds转座纯合类型,其余为Ds转座杂合类型,三者之间的比率同样符合1∶1∶2。根据上述结果可以认为,该突变体的产生是由于转座的Ds插入所引起的,遗传行为符合1对不完全显性基因的分离模式,表明该突变表型与Ds插入共分离,与F2群体颖果表型调查结果相吻合。
图2
新窗口打开|下载原图ZIP|生成PPT图2水稻pex1突变体表型及其分子检测
A：水稻pex1突变体谷粒和颖果表型。WT：野生型;pex1/+：突变杂合体;pex1/pex1：突变纯合体。B：Ds转座的PCR检测。M：100 bp DNA Ladder;1~7：检测植株,其中植株4、7的Ds未发生转座,仅存在FDS,1和6为Ds转座纯合体,仅存在EDS;2, 3, 5为Ds转座杂合体,既存在EDS又存在FDS。C：Ds插入突变体TAIL-PCR产物琼脂糖电泳分析。M：100 bp DNA Ladder; Ⅱ, Ⅲ：分别为TAIL-PCR第二、三轮产物。
Fig. 2Phenotypes of the pex1 mutant and its molecular detection
3 实践教学中的课堂组织方式
3.1 以科研思路为导向,创新实验内容、教学方法与组织方式
本实验作为分子遗传学综合性实验项目以培养学生创新思维与方法作为教学目标,将研究中发现的水稻Ds插入粒型突变体作为实验材料,以遗传分析和PCR检测技术为实操学习重点,并通过以下4步来完成实验教学：(1)引导学生发现问题;(2)通过表型调查和方差分析分析问题;(3)将表型观测与PCR检测技术相结合,对突变体进行鉴定与遗传分析提出问题;(4)通过知识拓展环节,结合转座因子内容,以该材料为例深入讲解转座子的特征及应用价值,加深学生对跳跃基因概念的理解,进一步研究问题。本实验可分4次课完成,每周1次课,每次课4个学时,分为以下3大部分：
(1)遗传分析实验部分1次课。1~2学时：首先提供给学生野生型突变体谷粒材料,引导学生对谷壳和米粒进行仔细观察并发现问题。教师再讲解考种的方法,指导学生用统计方法研究突变体表型特征。3~4学时：提供给每组F2群体谷粒,要求学生测量谷粒表型,利用卡方检验对突变体进行遗传分析。在该节课中,所有学生均表现出对实验的浓厚兴趣,并能按时完成实验。
(2) Ds转座的PCR检测部分2次课,第一次课的实验内容为提取水稻叶片DNA;第二次课的实验内容为PCR扩增及电泳,拍照记录带型,要求学生课后对实验结果进行统计分析。该部分实验内容相对复杂,会有个别学生需要通过重复实验才能完成。
(3)实验结果讨论与教学延伸部分1次课。首先组织学生汇报实验结果,了解学生对科学研究的思路、方法及结果分析的掌握情况。其次,教师结合理论知识讲解转座子对其他基因表达调控及其在基因组进化和基因功能研究方面的重要应用价值。并以pex1突变体为例,与学生探讨DS插入突变体的基因克隆技术和功能分析方法,拓展学生遗传知识的深度与宽度。通过对实验结果的讨论不仅有助于提升学生实验总结能力,更能激发学生进一步探索的兴趣,课后一般会有超过半数的同学主动联系老师进行研究性实验或创新训练项目拓展。
3.2 引导学生进行研究性实验
针对部分对该突变材料和基因克隆感兴趣的同学,引导他们利用开放实验平台,自行开展pex1材料的基因克隆研究。教学方法为：首先老师与学生通过课堂讨论和文献资料查阅了解插入突变体材料的基因克隆方法,与基因的图位克隆方法相比较,并设计实验方案。图位克隆又称定位克隆,用该方法分离基因的实质是寻找与目的基因紧密连锁的分子标记,无需预先知道基因的DNA顺序,但因需要构建遗传分离群体而耗时较长。就插入突变体而言,由于插入序列是已知的,可以作为插入位点的分子标签采用其他途径克隆目的基因。在此基础上,要求学生进一步查阅资料、阅读文献拿出插入突变体基因克隆可供选择的多种方案(例如反向PCR和TAIL-PCR等),通过比较分析,选用其中一种方法开展研究。在此过程中,教师需及时跟进、解疑答惑,并做好引导。教学实践表明,几乎所有学生均会优先考虑选用TAIL-PCR方法进行该突变体的基因克隆。
确定采用TAIL-PCR方法后,师生共同讨论、制定并修改完善实验方案,根据实验方案开展基因克隆研究,实验过程中教师实时跟进指导。虽然TAIL-PCR扩增核酸的基本原理与普通PCR无异,但是需要研究者对基本的PCR扩增原理和影响因素有深刻的理解和把握。TAIL-PCR需3轮扩增,前一轮产物稀释后用作后一轮扩增的模板,而且每轮的扩增尤其第一轮PCR扩增程序比普通PCR复杂;同时TAIL-PCR引物又有退火温度较高的嵌套式特异性引物和退火温度较低的兼并引物之别(图1)。这些都是有别于普通PCR的地方,也是该研究型实验教学的重点和难点。
TAIL-PCR 扩增Ds 3′旁邻序列如图2C所示,特异扩增的第三轮PCR产物可直接用于测序。 获得测序结果后,可指导学生利用公共核酸数据库(National Center for Biotechnology Information)进行初步的生物信息学分析,例如BLAST比对等便可获取目的基因的基本信息。本例的目的基因PEX1位于水稻第11染色体,属于类伸展蛋白基因家族。至此,插入突变体基因分离最关键的一步已完成。但是在实际教学中发现大多数学生需要重复多次才能获得理想扩增,在此过程中老师耐心指导尤为重要。实验最后,老师可结合TAIL-PCR电泳结果(如图2C)提出如何判断TAIL-PCR产物是否为特异扩增产物(由于特异引物是嵌套式的,第三轮产物比第二轮产物要小,可据此判别是否为特异扩增产物),分析研究结果及问题,撰写研究论文及研究报告。当然,后续有关PEX1基因功能的研究还有很多方面可以引导学生作进一步的思考。
4 教学中的思考与探索
该综合性实验融合了种子科学、经典遗传学的孟德尔遗传规律、基因组学的转座因子和统计学方差分析等理论知识内容,结合了表型观测、PCR检测技术、TAIL-PCR技术、基因克隆技术和方差分析法等实验内容。该综合性实验运用“综合性实验-研究性实验”的递进式教学体系,融合了“问题引导式—传授式—分组合作式—讨论分析式”混合教学法[14,15],以培养学生创新思维和方法为教学目标,让学生学会如何在科学研究中发现问题、分析问题、提出问题、研究问题,最后得出科学研究结论;让学生明白知识之间的联系性和整体性,进而实现知识融会贯通;有助于学生在团队协作中提升解决问题和创新实践的能力,这也是科研成果转化为本科实验教学的一个有益探索。
但在教学实践中,要使本课程达到最佳教学效果,还需要思考以下两点：首先,本实验所使用的材料来源于科学研究,与其相关的理论背景知识涉及不完全显性遗传与基因突变中的转座因子,然而这部分理论知识在遗传学理论教学中不是重点讲解内容,容易被学生忽视。因此在实验教学的过程中,需要将这部分理论知识整合进去,不仅让学生通过实验可以理解,更希望能引导学生将显性与不完全显性遗传、跳跃基因与转座子、转座因子的分类及其与基因突变进行对比分析,并将它们融会贯通;其次,综合性实验和研究性实验在教学中是层层递进式关系,在培养学生综合创新能力上发挥着不可替代的作用。综合性实验是学生进入科学研究领域前的传送带,旨在激发学生的研究兴趣,并使其掌握基本的研究方法和实验技能。研究性实验是学生进入科学研究领域的缩影,旨在让学生经历科学研究的过程,了解科学研究方法,进而培养学生创新思维及解决问题的能力。在综合性实验设计与教学过程中,如何加强综合性实验与研究性实验的关联性与整体性,激发学生研究的兴趣,引导学生进入研究性实验,一直是我们教学过程中需要不断思考与创新的地方。
参考文献 原文顺序
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被引期刊影响因子
DOI:10.16288/j.yczz.18-171URLPMID:30369473 [本文引用: 1]
Chinese genetics educators have carried out a comprehensive and systematic exploration and reform since 1978. With the guidance and help of the Genetics Society of China, they have made significant strides in the fields of genetics teaching system, publication of genetics textbooks, content of genetics teaching, workshop on genetics teaching, experimental teaching, application of advanced techniques, etc. These efforts have made remarkable achievements and promoted the vitality of genetics. The comprehensive development of education and teaching has trained a large number of excellent genetic talents for the development of China's economy and society. Here, we sum up the overall achievements of the teaching reform and propose some suggestions on the future development of genetics teaching in China, hoping that the quality of genetics teaching in China will take a new step in the new era.
DOI:10.16288/j.yczz.18-171URLPMID:30369473 [本文引用: 1]
Chinese genetics educators have carried out a comprehensive and systematic exploration and reform since 1978. With the guidance and help of the Genetics Society of China, they have made significant strides in the fields of genetics teaching system, publication of genetics textbooks, content of genetics teaching, workshop on genetics teaching, experimental teaching, application of advanced techniques, etc. These efforts have made remarkable achievements and promoted the vitality of genetics. The comprehensive development of education and teaching has trained a large number of excellent genetic talents for the development of China's economy and society. Here, we sum up the overall achievements of the teaching reform and propose some suggestions on the future development of genetics teaching in China, hoping that the quality of genetics teaching in China will take a new step in the new era.
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DOI:10.1038/cr.2012.151URL [本文引用: 1]
Increased crop yields are required to support rapid population growth worldwide. Grain weight is a key component of rice yield, but the underlying molecular mechanisms that control it remain elusive. Here, we report the cloning and characterization of a new quantitative trait locus (QTL) for the control of rice grain length, weight and yield. This locus, GL3.1, encodes a protein phosphatase kelch (PPKL) family - Ser/Thr phosphatase. GL3.1 is a member of the large grain WY3 variety, which is associated with weaker dephosphorylation activity than the small grain FAZ1 variety. GL3.1-WY3 influences protein phosphorylation in the spikelet to accelerate cell division, thereby resulting in longer grains and higher yields. Further studies have shown that GL3.1 directly dephosphorylates its substrate, Cyclin-T1; 3, which has only been rarely studied in plants. The downregulation of Cyclin-T1; 3 in rice resulted in a shorter grain, which indicates a novel function for Cyclin-T in cell cycle regulation. Our findings suggest a new mechanism for the regulation of grain size and yield that is driven through a novel phosphatase-mediated process that affects the phosphorylation of Cyclin-T1; 3 during cell cycle progression, and thus provide new insight into the mechanisms underlying crop seed development. We bred a new variety containing the natural GL3.1 allele that demonstrated increased grain yield, which indicates that GL3.1 is a powerful tool for breeding high-yield crops.
DOI:10.1186/1939-8433-5-4URLPMID:24764504 [本文引用: 1]
Seed size is an important trait in determinant of rice seed quality and yield. In this study, we report a novel semi-dominant mutant Small and round seed 5 (Srs5) that encodes alpha-tubulin protein. Lemma cell length was reduced in Srs5 compared with that of the wild-type. Mutants defective in the G-protein alpha subunit (d1-1) and brassinosteroid receptor, BRI1 (d61-2) also exhibited short seed phenotypes, the former due to impaired cell numbers and the latter due to impaired cell length. Seeds of the double mutant of Srs5 and d61-2 were smaller than those of Srs5 or d61-2. Furthermore, SRS5 and BRI1 genes were highly expressed in Srs5 and d61-2 mutants. These data indicate that SRS5 independently regulates cell elongation of the brassinosteroid signal transduction pathway.
DOI:10.1111/nph.12231URLPMID:23551229 [本文引用: 1]
· In order to understand the molecular genetic mechanisms of rice (Oryza sativa) organ development, we studied the narrow leaf2 narrow leaf3 (nal2 nal3; hereafter nal2/3) double mutant, which produces narrow-curly leaves, more tillers, fewer lateral roots, opened spikelets and narrow-thin grains. · We found that narrow-curly leaves resulted mainly from reduced lateral-axis outgrowth with fewer longitudinal veins and more, larger bulliform cells. Opened spikelets, possibly caused by marginal deformity in the lemma, gave rise to narrow-thin grains. · Map-based cloning revealed that NAL2 and NAL3 are paralogs that encode an identical OsWOX3A (OsNS) transcriptional activator, homologous to NARROW SHEATH1 (NS1) and NS2 in maize and PRESSED FLOWER in Arabidopsis. · OsWOX3A is expressed in the vascular tissues of various organs, where nal2/3 mutant phenotypes were displayed. Expression levels of several leaf development-associated genes were altered in nal2/3, and auxin transport-related genes were significantly changed, leading to pin mutant-like phenotypes such as more tillers and fewer lateral roots. OsWOX3A is involved in organ development in rice, lateral-axis outgrowth and vascular patterning in leaves, lemma and palea morphogenesis in spikelets, and development of tillers and lateral roots.
DOI:10.1104/pp.114.255737URLPMID:25855537 [本文引用: 1]
The methylation of histone H3 lysine 36 (H3K36) plays critical roles in brassinosteroid (BR)-related processes and is involved in controlling flowering time in rice (Oryza sativa). Although enzymes that catalyze this methylation reaction have been described, little is known about the recognition mechanisms to decipher H3K36 methylation information in rice. In this study, biochemical characterizations showed that MORF-RELATED GENE702 (MRG702) binds to trimethylated H3K4 and H3K36 (H3K4me3 and H3K36me3) in vitro. Similar to the loss-of-function mutants of the rice H3K36 methyltransferase gene SET DOMAIN GROUP725 (SDG725), the MRG702 knockdown mutants displayed typical BR-deficient mutant and late-flowering phenotypes. Gene transcription analyses showed that MRG702 knockdown resulted in the down-regulation of BR-related genes, including DWARF11, BRASSINOSTEROD INSENSITIVE1, and BRASSINOSTEROID UPREGULATED1, and several flowering genes, including Early heading date1 (Ehd1), Ehd2, Ehd3, OsMADS50, Heading date 3a, and RICE FLOWERING LOCUS T1. A binding analysis showed that MRG702 directly binds to the chromatin at target gene loci. This binding is dependent on the level of trimethylated H3K36, which is mediated by SDG725. Together, our results demonstrate that MRG702 acts as a reader protein of H3K4me3 and H3K36me3 and deciphers the H3K36 methylation information set by SDG725. Therefore, the role of MRG702 in the BR pathway and in controlling flowering time in rice is to function as a reader protein to decipher methylation information.
DOI:10.1016/0888-7543(95)80010-jURLPMID:7759102 [本文引用: 2]
Isolation of DNA segments adjacent to known sequences is a tedious task in genome-related research. We have developed an efficient PCR strategy that overcomes the shortcomings of existing methods and can be automated. This strategy, thermal asymmetric interlaced (TAIL)-PCR, utilizes nested sequence-specific primers together with a shorter arbitrary degenerate primer so that the relative amplification efficiencies of specific and nonspecific products can be thermally controlled. One low-stringency PCR cycle is carried out to create annealing site(s) adapted for the arbitrary primer within the unknown target sequence bordering the known segment. This sequence is then preferentially and geometrically amplified over nontarget ones by interspersion of high-stringency PCR cycles with reduced-stringency PCR cycles. We have exploited the efficiency of this method to expedite amplification and sequencing of insert end segments from P1 and YAC clones for chromosome walking. In this study we present protocols that are amenable to automation of amplification and sequencing of insert end sequences directly from cells of P1 and YAC clones.
DOI:10.1007/s00122-003-1416-8URLPMID:14513217 [本文引用: 4]
A collection of transposon Ac/ Ds enhancer trap lines is being developed in rice that will contribute to the development of a rice mutation machine for the functional analysis of rice genes. Molecular analyses revealed high transpositional activity in early generations, with 62% of the T0 primary transformants and more than 90% of their T1 progeny lines showing ongoing active transposition. About 10% of the lines displayed amplification of the Ds copy number. However, inactivation of Ds seemed to occur in about 70% of the T2 families and in the T3 generation. Southern blot analyses revealed a high frequency of germinal insertions inherited in the T1 progeny plants, and transmitted preferentially over the many other somatic inserts to later generations. The sequencing of Ds flanking sites in subsets of T1 plants indicated the independence of insertions in different T1 families originating from the same T0 line. Almost 80% of the insertion sites isolated showing homology to the sequenced genome, resided in genes or within a range at which neighbouring genes could be revealed by enhancer trapping. A strategy involving the propagation of a large number of T0 and T1 independent lines is being pursued to ensure the recovery of a maximum number of independent insertions in later generations. The inactive T2 and T3 lines produced will then provide a collection of stable insertions to be used in reverse genetics experiments. The preferential insertion of Ds in gene-rich regions and the use of lines containing multiple Ds transposons will enable the production of a large population of inserts in a smaller number of plants. Additional features provided by the presence of lox sites for site-specific recombination, or the use of different transposase sources and selectable markers, are discussed.
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DOI:10.1016/s0168-9452(01)00414-9URLPMID:11448751 [本文引用: 1]
Rice, with its small genome size and well-characterized molecular information, is an ideal model plant for cereal genomics research. Sequence of the rice (Oryza sativa) genome will be determined by the International Rice Genome Sequencing Project (IRGSP) in the near future. Therefore, a large population of mutant plants should be required for adequately assigning function to the abundant sequence information. Here we summarize strategies as well as the progress that has been made in producing gene tags that may be invaluable for understanding the functional genomics of rice.
DOI:10.1007/s00425-007-0576-1URL [本文引用: 1]
OSH6 (Oryza sativa Homeobox6) is an ortholog of lg3 (Liguleless3) in maize. We generated a novel allele, termed OSH6-Ds, by inserting a defective Ds element into the third exon of OSH6, which resulted in a truncated OSH6 mRNA. The truncated mRNA was expressed ectopically in leaf tissues and encoded the N-terminal region of OSH6, which includes the KNOX1 and partial KNOX2 subdomains. This recessive mutant showed outgrowth of bracts or produced leaves at the basal node of the panicle. These phenotypes distinguished it from the OSH6 transgene whose ectopic expression led to a “blade to sheath transformation” phenotype at the midrib region of leaves, similar to that seen in dominant Lg3 mutants. Expression of a similar truncated OSH6 cDNA from the 35S promoter (35S::ΔOSH6) confirmed that the ectopic expression of this product was responsible for the aberrant bract development. These data suggest that OSH6-Ds interferes with a developmental mechanism involved in bract differentiation, especially at the basal nodes of panicles.
DOI:10.1007/s00299-006-0259-6URL [本文引用: 1]
The shoot apical meristem (SAM) produces lateral organs in a regular spacing (phyllotaxy) and at a regular interval (phyllochron) during the vegetative phase. In a Dissociation (Ds) insertion rice population, we identified a mutant, compact shoot and leafy head 1 (csl1), which produced massive number of leaves (∼70) during the vegetative phase. In csl1, the transition from the vegetative to the reproductive phase was delayed by about 2 months under long-day conditions. With a reduced leaf size and severe dwarfism, csl1 failed to produce a normal panicle after the transition to reproductive growth. Instead, it produced a leafy panicle, in which all primary rachis-branches were converted to vegetative shoots. Phenotypically csl1 resembled pla mutants in short plastochron but was more severe in the conversion of the reproductive organs to vegetative organs. In addition, neither the expression nor the coding region of PLA1 or PLA2 was affected in csl1. csl1 is most likely a dominant mutation because no mutant segregant was observed in progeny of 67 siblings of the csl1 mutant. CSL1 may represent a novel gene, which functions downstream of PLA1 and/or PLA2, or alternatively functions in a separate pathway, involved in the regulation of leaf initiation and developmental transition via plant hormones or other mobile signals.
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