李富^,, 王延周, 严理, 朱四元, 刘头明^,*农业农村部麻类生物学与加工重点实验室/中国农业科学院麻类研究所, 湖南长沙 410205

Characterization of the expression profiling of circRNAs in the barks of stems in ramie

LI Fu^,, WANG Yan-Zhou, YAN Li, ZHU Si-Yuan, LIU Tou-Ming^,*Key Laboratory of Biological and Processing for Bast Fiber Crops, Ministry of Agriculture and Rural Affairs/Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, Hunan, China

通讯作者: *刘头明, E-mail:liutouming@caas.cn

收稿日期:2020-02-24接受日期:2020-07-2网络出版日期:2021-06-12

基金资助:

国家自然科学基金项目.31871678 中国农业科学院科技创新工程项目.CAAS-ASTIP-IBFC

Received:2020-02-24Accepted:2020-07-2Online:2021-06-12

Fund supported:

The National Natural Science Foundation of China.31871678 The Agricultural Science and Technology Innovation Program of China.CAAS-ASTIP-IBFC

作者简介 About authors
E-mail: 704510340@qq.com







摘要
苎麻[Boehmeria nivea (L.) Gaud.]是我国特色的天然纤维作物, 其纤维具有拉伸力强、纤维束长等特点。解析苎麻纤维发育调控机制, 对实现纤维产量和品质性状遗传改良具有重要意义。本研究基于高通量测序技术开展了苎麻茎顶部和中部的茎皮组织环状RNA (circRNA)分析, 在2个组织中共鉴定到5268个苎麻circRNA。比较circRNA的表达水平发现, 78个circRNA在2个组织中呈现差异表达。因苎麻茎中部韧皮纤维处于生长发育期, 而茎顶部韧皮纤维尚未起始生长, 推测这78个circRNA是纤维不同发育时期差异表达基因, 它们可能参与了苎麻的纤维发育调控。研究结果将为解析circRNA调控苎麻纤维生长发育机制奠定基础。
关键词： 苎麻;纤维发育;环状RNA;差异表达

Abstract
Ramie [Boehmeria nivea (L.) Gaud.] is a special natural fiber crops in China, and its fiber has many excellent characteristics, including long strands and well tensile strength. Elucidation of the mechanism for fiber formation will be helpful for the improvement of fiber yield and quality in ramie. In this study, the expressed analysis of circular RNA (circRNA) for the tissues of barks from the top stems and middle stems were performed by Illumina sequencing, respectively. The total of 5268 circRNAs were identified. Among these circRNAs, 78 showed differential expression between two examined tissues. Previous cytological observation suggested that the secondary cellular walls (SCWs) of fiber cells from the top of the ramie stems did not initiate growth and those from middle part of the ramie stems were thickening. Therefore, we speculated that these 78 differentially expressed circRNAs were potentially involved in the fiber development in ramie. The results provide an important basis for understanding the role of circRNA in the regulation of fiber development.
Keywords：ramie;fiber development;circRNA;differential expression


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本文引用格式
李富, 王延周, 严理, 朱四元, 刘头明. 苎麻茎皮环状RNA表达谱分析[J]. 作物学报, 2021, 47(6): 1020-1030. doi:10.3724/SP.J.1006.2021.04042
LI Fu, WANG Yan-Zhou, YAN Li, ZHU Si-Yuan, LIU Tou-Ming. Characterization of the expression profiling of circRNAs in the barks of stems in ramie[J]. Acta Agronomica Sinica, 2021, 47(6): 1020-1030. doi:10.3724/SP.J.1006.2021.04042


苎麻[Boehmeria nivea (L.) Gaud.]是我国重要的天然纤维作物, 有着悠久的栽培和加工历史, 其纤维及纤维织品是我国重要的工业原料和传统的出口创汇产品。中国作为苎麻的主产国, 其种植面积占全世界90%以上, 因此在国际上苎麻又被称为“中国草”。苎麻纤维是一种韧皮纤维, 具有拉伸力强、纤维长等特点, 其单纤维最长可达55 cm ^[1], 主要成分为纤维素、半纤维素和木质素。

目前, 苎麻中已有大量可能与纤维发育相关的基因被鉴定, 这为深入解析其纤维形成机制奠定了基础。基于RACE技术, 多个苎麻纤维素合成酶基因(CesA)已被鉴定和克隆^[2,3,4]。木质素作为纤维主要成分之一, 其多个生物合成酶基因, 如肉桂酸4-羟化酶(C4H)、香豆酸4-香豆酸:辅酶A连接酶(4CL)、肉桂酰辅酶a还原酶基因(CCR)也被鉴定和克隆^[5,6,7], 并发现它们的表达水平与苎麻纤维木质素含量显著相关^[8]。此外, 近10年来高通量测序技术快速发展, 也被广泛应用于苎麻纤维发育机制研究。Liu等^[9]分析苎麻转录组共获得36个在韧皮部中高表达的苎麻CesA基因。Chen等^[10]比较不同发育时期的苎麻韧皮组织转录组表达谱, 共鉴定到780个差异表达的基因, 其中含4个CesA基因、3个扩张蛋白基因、6个木聚糖水解酶基因, 它们很可能与苎麻的纤维发育相关。

非编码RNA是一类直接发挥催化和调控功能的转录本, 包含miRNA、长链非编码RNA (lncRNA)和环状RNA (circRNA)等^[11]。Wang等^[12]分析了苎麻纤维在4个不同发育时期的miRNA表达谱, 并在纤维伸长期和次生壁加厚期分别鉴定到150个和148个miRNA, 其中51个miRNA在纤维不同发育期呈现差异表达, 推测非编码RNA可能参与苎麻的纤维发育。circRNA是一类由mRNA前体经反向可变剪切而来的共价闭合且保守的环状转录本, 根据剪接位点可分为exonic circRNAs、intronic circRNAs、exonic-intronic circRNAs和intergenic circRNAs, 它们大多数具有较低的表达量, 且呈现组织特异性和细胞特异性表达。circRNA已知的功能包括隔离miRNA或蛋白质、转录调节和干扰可变剪接, 甚至翻译产生多肽^[13]。植物circRNA主要集中于生长发育及生物和非生物逆境研究^{[14,15,16,17,18,19]}, 如Zhang等^[14]在玉米和拟南芥中分别鉴定出2174个和1354个干旱相关的circRNA; Zuo等^[15]鉴定出番茄163个冷胁迫响应的circRNA; Wang等^[16]在野生型和有义/反义LeERF1转基因番茄果实中发现282个显著差异表达的cicrRNA; Wang等^[18]在小麦中发现62个circRNA响应干旱逆境; Gao等^[19]在葡萄中鉴定了475个响应寒冷的差异表达circRNA。尽管circRNA被发现广泛参与了植物的生长调控, 但有关它在植物纤维发育中的功能角色, 当前仍鲜有研究。本研究将开展苎麻纤维发育期的全基因组circRNA表达谱分析, 并筛选差异表达的circRNA, 旨在为理解circRNA在植物纤维发育中的调控角色奠定基础。

1 材料与方法

1.1 试验材料和取样

本试验以中苎1号为研究材料。2016年, 将中苎1号扦插苗栽种于中国农业科学院麻类研究所沅江田间试验基地。经切片显微观察, 茎中部韧皮纤维处于生长期, 次生壁正在加厚; 而茎顶部韧皮纤维尚未起始生长^[10]。因此, 在2018年5月, 当具有两年龄的苎麻长到约1.5 m高时, 按照Chen等^[10]方法, 分别取茎中部(the barks sample collected from the middle, MPS)和顶部(the barks sample collected from the top, TPS)的茎皮组织(图1)。对3株苎麻单独取样, 分别作为3个试验重复。组织样品经液氮速冻后置于-80℃低温保存, 以备后续试验使用。

图1

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图1苎麻circRNA测序取样部位示意图

MPS和TPS分别指从茎中部和顶部收集的茎皮组织样品, 其中位于茎顶端的纤维细胞次生壁尚未加厚, 而处于茎中部的纤维细胞次生壁显著加厚(图片引自Chen等^[10])。
Fig. 1A picture shows the sampling position for circRNA sequencing

MPS and TPS represent the barks sample collected from the middle and top stems, respectively. This picture displays that the secondary cellular walls (SCWs) of fiber cells from the top of the ramie stems do not initiate growth and those from middle part of the ramie stems are thickening. This figure is cited from the study of Chen et al.^[10]

1.2 文库构建和高通量测序

利用植物RNA提取试剂TRIzol (Invitrogen, CA, USA)提取6个样品的总RNA。之后对6个RNA样品单独构建circRNA测序文库, 具体流程如下: 使用试剂盒(Ribo-Zero Gold rRNA Removal Kit, Illumina)去除total RNA中的核糖体RNA, 并使用Agilent 2100Bioanalyzer (Agilent Technologies)验证纯化效果; RNaseR反应体系(TruSeq Stranded Total RNA with Ribo-Zero Plant, Illumina, USA)消化线性RNA, 将反应产物纯化, 并打断使其片段化; 以片段化环状RNA为模板, 使用反转录试剂盒(SuperScript III First-Strand Synthesis System for RT-PCR, Invitrogen)先后合成第一链cDNA和第二链cDNA, 并使用试剂盒(PureLink Quick Gel Extraction and PCR Purification Combo Kit, Invitrogen)纯化回收、黏性末端修复、cDNA的3°末端加上碱基“A”并连接接头, 最后进行PCR扩增; 构建好的文库用Agilent2100 Bioanalyzer (Agilent Technologies)和StepOnePlus Real-time PCR System (ABI)质检, 合格后的文库则通过Illumina测序平台(HiSeq 2500, Illumina, USA)进行高通量测序, 从而获得原始的序列reads。

1.3 circRNA预测

对原始序列进行过滤, 主要包括去除接头污染的序列reads、未知碱基N含量大于5%的reads及低质量的序列reads, reads中超过20%的碱基为质量值低于30的碱基(即碱基错误率大于0.032), 从而获得高质量的clean reads。将clean reads比对到苎麻参考基因组上^[20], 然后通过CIRI^[21]、find_circ ^[22] 2个软件预测circRNA, 并且依据circRNA起始、终止位置来整合2个软件的预测结果。若2个circRNA的起始和终止位置均相近(10个碱基以内), 则将这2个circRNA合并为1个。

1.4 circRNA表达定量及差异表达分析

本研究根据比对至circRNA两端的junction reads数来对circRNA表达定量^[23]。因circRNA是由CIRI和find_circ 2个软件预测而来, 故就每个circRNA, 我们计算2个软件注释的junction reads数的平均值, 将其作为该circRNA最终的junction reads数。采用RPB (junction reads per billion mapped reads, 即比对上基因组的所有reads标准化到十亿后跨过backspliced位点的junction reads数目)对各样品作均一化处理。原始的clean reads序列及其表达定量已被提交到DDBJ/EMBL/GenBank数据库(序列号位GSE130587)。

利用DEseq2软件^[24]筛选差异表达的circRNA。利用DEseq2软件计算circRNA在茎顶部和茎中部的表达量差异倍数和P值, 通过Benjamini-Hochberg统计方法^[25]对P值进行校正。对于每个circRNA, 若其校正的P值小于0.05, 表达差异大于2倍, 则认为该circRNA在2个组织中存在表达差异。

1.5 荧光定量PCR (qRT-PCR)分析

分别提取茎顶部和茎中部RNA (3个生物学重复, 共6个样品), 用DNase I (Fermentas, Canada)进行消化处理, 去除其中的DNA。之后, 利用M-MuLV反转录试剂盒(Fermentas)合成第1链cDNA。使用iTaq Universal SYBR Green super mix试剂盒(Bio-Rad, USA)配置qRT-PCR反应体系, 并在iQ5实时荧光PCR仪上进行PCR。PCR反应程序: 第一步95℃ 30 s; 第二步40个循环: 95℃ 15 s, 60 ℃ 30 s。就每个circRNA, 每个样品进行5个PCR反应重复。18S核糖体RNA在植物各器官中呈现组成性表达, 因此被用作内参基因^[12]。circRNA和内参基因引物见表1。依据Livak的2^-ΔΔCT方法^[26]计算各circRNA在2个组织中的相对表达水平和标准误。

Table 1
表1
表1用于qRT-PCR分析的苎麻差异表达circRNA和内参基因引物序列
Table 1Primer sequence of differentially expressed circRNAs and the control gene for qRT-PCR in ramie

circRNA名称 circRNA ID	正向引物 Forward primer (5°-3°)	反向引物 Reverse primer (5°-3°)
scaffold270:40371|40783	TGGCATGAATCACCGAACCTG	TGTCATGACGCGCCCATTCCT
scaffold6:1851520|1851960	ATGAAATCAGGGAATGCCAA	CTCCCAAACTTCACCGTC
scaffold360:24869|25462	CTTTCCTTGCTTCGCCAAC	TTCCCTGCTACTCTATTGCT
scaffold360:11517|11838	TCCACTAGCGTCATTCGAAG	CTAGGTCAGAAAAGCGAGAAC
scaffold36:1536277|1547559	TTGATCTTGGCCATGTCCCGA	ACGGTTCAATAAGGCGAGA
scaffold110:48151|61235	AGAATCCACAATACCGTCAGC	TTGTGTATTCATTTGGCCCAT
scaffold30:600099|602644	ATAAAGAGTCGTTGGGCCTT	TGAACTTCCTTTCCGCCAGA
scaffold11:2168138|2169190	GTCCACAGAGCGATCATATGTC	TTTTAGGCACTTACCGGGTT
scaffold360:15097|17687	CGTTTGACTTGATCCCGACAG	CAGAGCAAATGAGGGTATAGCAA
scaffold128:291760|298584	ACAAAAAGCAATGGCAATTACGG	AACAGACTCACCAATGGCATC
18S	ATGATAACTCGACGGATCGC	CTTGGATGTGGTAGCCGTTT

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2 结果与分析

2.1 circRNA的鉴定

经测序, 6个样品共产生大约6.46亿个原始序列reads, 经过滤后得到共计4.45亿个高质量的clean reads, 序列长度合计达到66.7 Gb (表2)。每个样品过滤后均有约74.0百万个clean reads, 且其Q30值均达到96%以上, 说明测序获得的数量和质量均足以开展下一步的circRNA分析。

Table 2
表2
表2苎麻6个茎皮组织样本的circRNA测序数据统计
Table 2Data statistics from circRNA sequencing for six samples of stem barks in ramie

样品 Sample	原始read数 Raw read number (Million)	Clean read数 Clean read number (Million)	总长度 Total length (Gb)	Q30值 Q30 value (%)	Clean reads产出率 Yielded ratio of clean reads (%)
MPS1	101.23	74.3	11.1	96.4	73.4
MPS2	106.02	74.5	11.2	96.0	70.3
MPS3	107.77	74.3	11.1	96.7	68.9
TPS1	102.76	74.0	11.1	96.0	72.0
TPS2	119.18	73.7	11.1	96.1	61.9
TPS3	109.41	73.7	11.1	96.4	67.4

MPS和TPS分别指从茎中部和顶部收集的茎皮组织样品; Q30值为序列过滤后的reads中质量值大于30的碱基数占总碱基数的比例。
MPS and TPS represent the barks sample collected from the middle and top stems, respectively; Q30 value indicates the ratio of bases with quality of more than 30 in the clean read.

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基于这些高质量的序列, 本研究在苎麻茎顶部和中部韧皮组织中共鉴定到5268个circRNA, 其中539个位于基因间(intergenic), 而4729个(89.8%)则位于基因内部(intragenic)。对这4729个基因进行GO (gene ontology)功能分类发现, 它们主要归类到结合(binding)、催化活性(catalytic activity)、细胞过程(cellular process)、代谢过程(metabolic process)、膜(membrane)等功能类别(Q < 0.01)(图2)。

图2

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图2可转录circRNA的4729个苎麻基因GO功能分类

Fig. 2GO functional classification of 4729 transcribed circRNAs genes in ramie

2.2 circRNA表达谱分析

显微观察显示, 苎麻茎中部韧皮纤维处于生长期, 次生壁正在加厚; 而茎顶部韧皮纤维尚未起始生长(图1)。比较5268个苎麻circRNA在2个茎韧皮部位中的表达水平发现, 相对于TPS, 在MPS韧皮中有77个circRNA显著上调表达, 仅1个circRNA表达水平下调(图3)。因茎中部和顶部的纤维处于不同发育时期, 说明这78个circRNA在2个不同纤维发育时期存在表达差异。在这78个circRNA中, 11个circRNA的表达水平差异倍数达到30倍以上(表3)。其中scaffold13:3497301|3498287和scaffold7:2328081|2328970在MPS中的表达分别上调了42.7倍和42.0倍, 它们分别定位在苎麻基因whole_GLEAN_10021135和whole_GLEAN_10025341中; whole_GLEAN_10021135编码贝壳杉烯酸氧化酶, whole_GLEAN_10025341则为酪蛋白激酶编码基因。此外, 在这78个差异表达的circRNA中, 74个(94.9%)位于基因内部, 其中scaffold1:2974233| 2974487被定位在一个knotted-1-like Homeobox基因(whole_GLEAN_10029667)中, 其上调表达倍数达到30.6。

图3

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图3苎麻MPS和TPS茎皮中差异表达circRNA鉴定

图中红色、蓝色和灰色的数据点分别代表上调表达、下调表达及无表达差异的circRNA; 横坐标和纵坐标分别代表某circRNA在TPS和MPS组织中的read数的对数值。缩写同表2。
Fig. 3Identification of differentially expressed circRNA by comparing gene expression level between TPS and MPS libraries

Red dots represent transcripts more prevalent in the MPS library, blue dots show those present at a lower frequency in the MPS, and gray dots indicate transcripts that did not change significantly; X and Y axes represent the logarithm of read number for each circRNA in TPS and MPS libraries, respectively. Abbreviations are the same as those given in Table 2.


Table 3
表3
表3苎麻MPS和TPS茎皮中差异表达circRNA信息表
Table 3Detail for differentially expressed circRNA identified between the bast barks of MPS and TPS

circRNA名称 circRNA ID	类型 Type	MPS read数 MPS-reads number	TPS read数 TPS-reads number	差异倍数 Differential fold (MPS/TPS)	调控方向 Direction regulated	校正P值 Adjusted P-value	对应基因 Gene located by circRNA	对应基因功能 Annotationof gene located by circRNA
scaffold13:3497301|3498287	Introgenic	553.7	13.0	42.7	上调 Up-regulated	2.6E-04	whole_GLEAN_10021135	Ent-kaurenoic acid oxidase 1
scaffold7:2328081|2328970	Iintrogenic	760.3	18.1	42.0	上调Up-regulated	2.8E-04	whole_GLEAN_10025341	Casein kinase 1
scaffold27:618745|619712	Introgenic	484.5	12.5	38.9	上调Up-regulated	3.9E-04	whole_GLEAN_10015712	Cleavage and polyadenylation specificity factor subunit 3-I
scaffold24:109942|110887	Introgenic	461.0	12.8	35.9	上调Up-regulated	5.4E-04	whole_GLEAN_10016716	Protein APEM9
scaffold68:19731|20827	Introgenic	433.3	12.1	35.7	上调Up-regulated	5.6E-04	whole_GLEAN_10010106	Protein CHROMATIN REMODELING 5
scaffold777:22008|22923	Introgenic	485.5	14.2	34.2	上调Up-regulated	6.7E-04	whole_GLEAN_10001638	Non-specific phospholipase C3
scaffold1:1651705|1652476	Introgenic	507.7	14.9	34.1	上调Up-regulated	6.7E-04	whole_GLEAN_10029483	Monogalactosyldiacylglycerol synthase
scaffold11:2168138|2169190	Intergenic	401.2	11.8	33.9	上调Up-regulated	6.8E-04
scaffold14:2019005|2020131	Introgenic	568.2	17.5	32.4	上调Up-regulated	8.2E-04	whole_GLEAN_10020529	Peroxisome biogenesis protein 1
scaffold1:2974233|2974487	Introgenic	418.1	13.7	30.6	上调Up-regulated	1.0E-03	whole_GLEAN_10029667	Homeobox protein knotted-1-like 1
scaffold4:2794969|2795547	Introgenic	328.8	10.8	30.5	上调Up-regulated	1.0E-03	whole_GLEAN_10027195	Lissencephaly-1 homolog
scaffold13:1482426|1482614	Introgenic	288.1	10.8	26.7	上调Up-regulated	1.7E-03	whole_GLEAN_10020870	Threonine-protein kinase PBL28
scaffold42:394825|395928	Introgenic	303.4	11.9	25.5	上调Up-regulated	2.0E-03	whole_GLEAN_10012793	U11/U12 small nuclear ribonucleoprotein 65 kDa protein
scaffold110:48151|61235	Introgenic	256.8	10.3	24.9	上调Up-regulated	2.2E-03	whole_GLEAN_10007543	Protein TIC 40
scaffold6:1851520|1851960	Introgenic	400.8	16.3	24.5	上调Up-regulated	2.3E-03	whole_GLEAN_10025870	DEAD-box ATP-dependent RNA helicase 50
scaffold6:319819|320593	Introgenic	239.6	10.5	22.9	上调Up-regulated	2.9E-03	whole_GLEAN_10025672	GBF-interacting protein 1
scaffold22:2080855|2081636	Introgenic	308.2	13.6	22.6	上调Up-regulated	3.1E-03	whole_GLEAN_10017734	SOK1 kinase belonging to the STE20/SPS1/GC kinase family
scaffold38:1007107|1007440	Introgenic	229.5	10.2	22.5	上调Up-regulated	3.1E-03	whole_GLEAN_10013247	Lipoxygenase 6
scaffold4:984097|984791	Introgenic	225.4	10.3	21.8	上调Up-regulated	3.5E-03	whole_GLEAN_10026982	V-type proton ATPase subunit a1
scaffold36:1130226|1130557	Introgenic	332.4	15.3	21.8	上调Up-regulated	3.5E-03	whole_GLEAN_10013939	Primary amine oxidase
scaffold9:3087645|3088126	Introgenic	261.8	12.2	21.5	上调Up-regulated	3.7E-03	whole_GLEAN_10024168	Transcription factor bHLH144
scaffold69:390306|390963	Introgenic	230.6	11.2	20.6	上调Up-regulated	4.2E-03	whole_GLEAN_10010042	Formin-like protein 16
scaffold244:78865|79334	Introgenic	216.4	10.9	19.9	上调Up-regulated	4.7E-03	whole_GLEAN_10004368	Uncharacterized mitochondrial protein ymf40
scaffold11:2226699|2233286	Introgenic	273.2	13.9	19.6	上调Up-regulated	4.9E-03	whole_GLEAN_10021948	Cold-regulated 413 plasma membrane protein 2
scaffold23:488065|489466	Introgenic	198.8	10.2	19.4	上调Up-regulated	5.1E-03	whole_GLEAN_10017916	Phosphoinositide phosphatase SAC1
scaffold59:336176|337027	Introgenic	192.9	10.1	19.1	上调Up-regulated	5.4E-03	whole_GLEAN_10011259	Glycosylphosphatidylinositol anchor synthesis protein
scaffold270:63772|64261	Introgenic	215.3	11.3	19.0	上调Up-regulated	5.5E-03	whole_GLEAN_10004135	ATP synthase protein YMF19
scaffold21:626345|627257	Introgenic	179.7	9.7	18.6	上调Up-regulated	5.9E-03	whole_GLEAN_10019902	Poly [ADP-ribose] polymerase 1
scaffold41:981509|982441	Introgenic	232.5	12.9	18.0	上调Up-regulated	6.5E-03	whole_GLEAN_10013030	WD40 repeat protein
scaffold25:1518597|1519148	Introgenic	183.3	10.3	17.7	上调Up-regulated	6.8E-03	whole_GLEAN_10016591	Protein LSD1
scaffold69:199217|199851	Introgenic	194.3	11.2	17.3	上调Up-regulated	7.4E-03	whole_GLEAN_10010022	U-box domain-containing protein 14
scaffold37:1378235|1379257	Introgenic	163.1	9.5	17.2	上调Up-regulated	7.5E-03	whole_GLEAN_10013509	Uncharacterized protein At1g51745
scaffold41:312471|314479	Introgenic	168.0	10.2	16.5	上调Up-regulated	8.5E-03	whole_GLEAN_10012948	Nucleolar GTPase 2
scaffold30:600099|602644	Introgenic	156.9	9.6	16.3	上调Up-regulated	8.9E-03	whole_GLEAN_10015039	Protein CROWDED NUCLEI 3
scaffold34:2347785|2349055	Introgenic	144.0	9.0	16.0	上调Up-regulated	9.3E-03	whole_GLEAN_10017394	B3 domain-containing protein07g0563300
scaffold360:24869|25462	Introgenic	174.8	11.0	15.9	上调Up-regulated	9.4E-03	whole_GLEAN_10003361	Cytochrome c oxidase subunit 2
scaffold1:5693805|5694897	Introgenic	169.3	10.6	15.9	上调Up-regulated	9.5E-03	whole_GLEAN_10030060	Internal alternative NAD(P)H-ubiquinone oxidoreductase A1
scaffold244:79500|79823	Introgenic	155.0	9.9	15.7	上调Up-regulated	9.8E-03	whole_GLEAN_10004368	Uncharacterized mitochondrial protein ymf40
scaffold270:63801|64263	Introgenic	155.3	10.1	15.4	上调Up-regulated	1.0E-02	whole_GLEAN_10004135	ATP synthase protein YMF19
scaffold112:248381|248869	Introgenic	148.3	9.6	15.4	上调Up-regulated	1.0E-02	whole_GLEAN_10007469	Peptidyl-prolyl cis-trans isomerase CYP71
scaffold36:1536277|1547559	Introgenic	150.7	9.9	15.2	上调Up-regulated	1.1E-02	whole_GLEAN_10013997	Cytochrome P450 71A1
scaffold360:12593|13058	Intergenic	155.4	10.4	15.0	上调Up-regulated	1.1E-02
scaffold1:142468|143089	Introgenic	128.5	9.0	14.3	上调Up-regulated	1.3E-02	whole_GLEAN_10029278	Protein Mut11
scaffold29:718451|719832	Introgenic	122.4	8.8	13.9	上调Up-regulated	1.4E-02	whole_GLEAN_10015276	E4 SUMO-protein ligase PIAL2
scaffold360:15097|17687	Introgenic	121.8	8.8	13.9	上调Up-regulated	1.4E-02	whole_GLEAN_10003361	Cytochrome c oxidase subunit 2
scaffold39:1205365|1205747	Intergenic	121.1	8.9	13.6	上调Up-regulated	1.5E-02
scaffold19:1034720|1049699	Introgenic	115.4	8.8	13.1	上调Up-regulated	1.6E-02	whole_GLEAN_10018479	Short-chain dehydrogenase/reductase 2b
scaffold27:1733501|1744156	Introgenic	102.4	8.0	12.8	上调Up-regulated	1.7E-02	whole_GLEAN_10015833	Large ribosomal RNA subunit accumulation protein YCED homolog 2
scaffold270:115352|115747	Introgenic	98.1	7.7	12.7	上调Up-regulated	1.8E-02	whole_GLEAN_10004138	Hypothetical protein
scaffold11:3427880|3429252	Introgenic	95.6	7.8	12.3	上调Up-regulated	2.0E-02	whole_GLEAN_10022104	Probable ubiquitin-like-specific protease 2B
scaffold5:2904984|2905988	Introgenic	96.1	7.9	12.1	上调Up-regulated	2.0E-02	whole_GLEAN_10026531	Transcription factor bHLH68
scaffold22:2902269|2903224	Introgenic	1019.2	88.3	11.5	上调Up-regulated	2.0E-02	whole_GLEAN_10017861	TATA-binding protein-associated factor BTAF1
scaffold41:1051261|1051835	Introgenic	333.0	30.0	11.1	上调Up-regulated	2.5E-02	whole_GLEAN_10013037	Probable E3 ubiquitin-protein ligase RHB1A
scaffold423:8362|8891	Introgenic	75.7	6.9	11.0	上调Up-regulated	2.6E-02	whole_GLEAN_10002921	NADH-ubiquinone oxidoreductase chain 2
scaffold50:971580|972301	Introgenic	75.7	6.9	11.0	上调Up-regulated	2.6E-02	whole_GLEAN_10022800	Uncharacterized conserved protein
scaffold29:144492|145002	Introgenic	1922.2	200.1	9.6	上调Up-regulated	2.6E-02	whole_GLEAN_10015203	A Plastidial ribosomal protein S1
scaffold423:7458|7843	Introgenic	69.4	6.5	10.6	上调Up-regulated	2.8E-02	whole_GLEAN_10002921	NADH-ubiquinone oxidoreductase chain 2
scaffold41:1051261|1051640	Introgenic	383.7	36.9	10.4	上调Up-regulated	3.0E-02	whole_GLEAN_10013037	Probable E3 ubiquitin-protein ligase RHB1A
scaffold128:291760|298584	Introgenic	244.6	25.7	9.5	上调Up-regulated	3.7E-02	whole_GLEAN_10007591	Histone-lysine N-methyltransferase ATXR6
scaffold42:575382|577290	Introgenic	248.1	26.2	9.5	上调Up-regulated	3.7E-02	whole_GLEAN_10012809	Uncharacterized conserved protein
scaffold4:4247567|4248126	Introgenic	238.8	25.3	9.4	上调Up-regulated	3.8E-02	whole_GLEAN_10027396	ALBINO3-like protein 2
scaffold26:228677|229118	Introgenic	233.3	25.6	9.1	上调Up-regulated	4.1E-02	whole_GLEAN_10016157	Protein BTR1
scaffold81:336505|338444	Introgenic	223.2	24.5	9.1	上调Up-regulated	4.1E-02	whole_GLEAN_10009183	Protein FORGETTER 1
scaffold8:474393|475508	Introgenic	633.7	72.5	8.7	上调Up-regulated	4.1E-02	whole_GLEAN_10024439	Uncharacterized protein At3g06530
scaffold3:3339987|3341286	Introgenic	246.4	27.5	8.9	上调Up-regulated	4.2E-02	whole_GLEAN_10028049	Aspartate aminotransferase 3
scaffold133:143853|144812	Introgenic	212.1	23.8	8.9	上调Up-regulated	4.3E-02	whole_GLEAN_10006440	Protein RNA-directed DNA methylation 3
scaffold16:2445618|2446250	Introgenic	203.6	23.3	8.7	上调Up-regulated	4.5E-02	whole_GLEAN_10019477	Alternative NAD(P)H-ubiquinone oxidoreductase C1
scaffold270:115352|115722	Introgenic	202.7	23.3	8.7	上调Up-regulated	4.5E-02	whole_GLEAN_10004138	Hypothetical protein
scaffold8:2674752|2676656	Introgenic	580.2	69.2	8.4	上调Up-regulated	4.6E-02	whole_GLEAN_10024738	COMPASS-like H3K4 histone methylase component WDR5A
scaffold360:11517|11838	Intergenic	560.1	68.9	8.1	上调Up-regulated	4.9E-02
scaffold12:2523138|2525034	Introgenic	529.5	65.4	8.1	上调Up-regulated	4.9E-02	whole_GLEAN_10021469	Tetraspanin-18
scaffold40:1450202|1450949	Introgenic	188.2	22.5	8.3	上调Up-regulated	5.0E-02	whole_GLEAN_10013727	Proline-rich receptor-like protein kinase PERK8
scaffold270:40371|40783	Introgenic	991.9	121.6	8.2	上调Up-regulated	5.0E-02	whole_GLEAN_10004133	Hypothetical protein
scaffold7:3133443|3134407	Introgenic	218.9	26.3	8.3	上调Up-regulated	5.0E-02	whole_GLEAN_10025435	DEAD-box ATP-dependent RNA helicase 13
scaffold69:125839|129078	Introgenic	195.2	23.4	8.3	上调Up-regulated	5.0E-02	whole_GLEAN_10010014	Exocyst complex component EXO70A1
scaffold38:37152|37816	Introgenic	181.8	22.1	8.2	上调Up-regulated	5.0E-02	whole_GLEAN_10013105	DNA replication licensing factor MCM2
scaffold9:1148862|1149242	Introgenic	196.3	24.4	8.0	上调Up-regulated	5.0E-02	whole_GLEAN_10023894	Pyrophosphate-energized vacuolar membrane proton pump
scaffold2:6347151|6348009	Introgenic	9.1	105.2	11.5	下调Down-regulated	2.3E-02	whole_GLEAN_10029246	N-Acetylglucosamine kinase

缩写同表2。
Abbreviations are the same as those given in Table 2.

新窗口打开|下载CSV

为了验证这些circRNA的差异表达结果, 本研究随机选取了10个circRNA, 通过qRT-PCR进一步分析它们在2个组织中的表达水平。由图4可知, 10个circRNA均呈现出差异表达, 且差异表达倍数明显高于测序分析结果, scaffold6:1851520| 1851960和scaffold30:600099|602644的差异表达倍数甚至达到上千倍。可见, 与高通量测序技术相比, qRT-PCR检测circRNA具有更高的灵敏度。说明这78个circRNA的差异表达是真实可靠的。

图4

新窗口打开|下载原图ZIP|生成PPT
图4qRT-PCR分析10个苎麻circRNA在MPS和TPS茎皮中的相对表达水平

缩写同表2。
Fig. 4Relative expression level of ten circRNAs stem bark tissues of MPS and TPS by qRT-PCR in ramie

Abbreviations are the same as those given in Table 2.

3 讨论

纤维广泛存在于植物各器官中, 具有支撑器官形态、植株直立生长, 及抵抗病原菌攻击等作用。此外, 植物纤维也是重要的工业原料, 广泛用于纺织、造纸等工业领域。可见, 不管是对植物本身, 还是对国民经济, 植物纤维具有非常重要的作用。目前, 有关植物纤维生物合成机制在模式植物拟南芥中研究的比较多, 发现其主要由一系列NAC (NAM, ATAF1/2和CUC2)和MYB (v-myb avian myloblastosis viral oncogene homolog)转录因子调控, 它们形成分层次的网络并逐级调控整个纤维发育过程^[27]。蛋白翻译后修饰如磷酸化、泛素化等也参与植物纤维的发育调控^[28,29,30]。此外, 非编码RNA也可参与植物的纤维发育调控, 如柑橘miR397a可以调控纤维发育控制基因LAC17和LAC4的表达^[31], 而拟南芥miR319则靶向调控TCP4基因的表达, 进而抑制纤维发育^[32]。

circRNA是一类重要的非编码RNA, 广泛参与花发育、果实成熟、逆境响应等多个生命过程^[33]。植物中存在大量的circRNA, 如PlantcircBase数据库^[34]已收录了40,311个水稻(Oryza sativa) circRNA、38,938个拟南芥(Arabidopsis thaliana) circRNA、6302个玉米(Zea mays) circRNA、7806个大豆(Glycine max) circRNA和1976个番茄(Solanum lycopersicum) circRNA。Wang等^[35]系统研究了244个玉米RNA-Seq样本和288个水稻RNA-Seq样本, 鉴定到38,785个玉米circRNA和63,048个水稻circRNA, 并发现在玉米中有11,206个circRNA响应干旱逆境, 6770个circRNA响应盐逆境, 而在水稻中有824、6313和5724个circRNA分别响应干旱、盐和冷逆境。Zhang等^[36]首次发现可能与花生种子发育相关的347个差异表达circRNA, 并通过qRT-PCR验证了其中15个circRNA的表达水平。茉莉酸甲酯(methyl jasmonic acid, MeJA)在植物生长和防御中发挥关键作用, 拟南芥种子用MeJA处理后, 经高通量测序鉴定出8588个circRNA, 其中有385个circRNA响应MeJA处理^[37]。已有研究表明, circRNA能够参与调控其宿主基因表达, 例如在水稻中过表达circRNA Os08circ16564降低其宿主基因AK064900在叶和花序中的表达^[37]。Gao等^[19]在低温胁迫下的葡萄叶片中鉴定了475个差异表达的circRNA, 通过异源过度表达一种源自甘油-3-P-酰基转移酶的circRNA (Vv-circATS1), 可以增强拟南芥(Arabidopsis thaliana)的耐寒性, 而源自同一序列的线性RNA则不能, 这为植物耐寒提供新见解。

当前对circRNA在植物纤维发育中的功能角色仍有待深入研究。本研究鉴定到78个苎麻circRNA在纤维发育不同时期呈现差异表达, 这将为研究circRNA在苎麻纤维发育中的功能奠定重要基础。在78个差异表达circRNA中, 有74个circRNA位于基因内部, 占比达94.9%, 这个比例高于苎麻总circRNA中该类型的比例(89.8%), 其表达是否影响相应基因的表达, 目前仍难以确定。例如knotted-1-like Homeobox家族是植物发育中重要的转录因子家族, 其多个成员, 如拟南芥KNAT7、棉花GhKNL1均被发现参与植物的纤维发育调控^[38,39]。此外, 在纤维发育过程中, 胞外复合体酶EXO70A1主要负责转运纤维素合成酶^[40], 而磷酸酶SAC1在纤维次生壁加厚过程中具有重要功能^[41]。本研究发现, whole_GLEAN_10029667、whole_GLEAN_10017916和whole_GLEAN_10010014分别注释为拟南芥纤维发育基因KNAT7、SAC1和EXO70A1的同源基因,circRNAscaffold1:2974233|2974487、scaffold23:488065| 489466和scaffold69:125839|129078分别定位到这3个苎麻基因中。因circRNA可通过miRNA海绵功能、干扰可变剪切、结合蛋白等方式调控相应基因表达, 这3个circRNA的上调表达或许会影响到相应的whole_GLEAN_10029667、whole_GLEAN_10017916和whole_GLEAN_10010014表达, 进而可能在纤维发育中发挥功能。因此, 本研究将为我们深入研究这3个circRNA和相应基因的功能及它们的表达调控关系奠定基础。

4 结论

本研究首次在苎麻中鉴定到5268个circRNA, 并发现其中78个circRNA在纤维发育的2个不同时期呈现差异表达。这些circRNA的鉴定为研究苎麻生长发育, 尤其是纤维的生长发育调控功能提供重要基础。

参考文献 View Option 原文顺序
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被引期刊影响因子

[1] Aldaba V. The structure and development of the cell wall in plants I. Bast fibers of Boehmeria and Linum
Am J Bot, 1927,14:16-24.
[本文引用: 1]

[2] 蒋杰, 揭雨成, 周清明, 周精华, 朱守晶, 邢虎成, 钟英丽. 苎麻纤维素合酶基因BnCesAl全长cDNA的克隆与表达分析
植物遗传资源学报, 2012,13:851-857.
[本文引用: 1]

Jiang J, Jie Y C, Zhou Q M, Zhou J H, Zhu S J, Xing H C, Zhong Y L. Full-length cDNA cloning and express analysis of BnCesA1 in ramie
J Plant Genet Resour, 2012,13:851-857 (in Chinese with English abstract).
[本文引用: 1]

[3] 刘昱翔, 陈建荣, 彭彦, 黄妤, 赵燕, 黄丽华, 郭清泉, 张学文. 两种苎麻纤维素合酶基因cDNA序列的克隆及表达
作物学报, 2014,40:1925-1935.
[本文引用: 1]

Liu Y X, Chen J R, Peng Y, Huang Y, Zhao Y, Huang L H, Guo Q Q, Zhang X W. cDNA cloning and expression of two cellulose synthase genes from Boehmerianivea
Acta Agron Sin, 2014,40:1925-1935 (in Chinese with English abstract).
[本文引用: 1]

[4] 田志坚, 易蓉, 陈建荣, 郭清泉, 张学文. 苎麻纤维素合成酶基因cDNA的克隆及表达分析
作物学报, 2008,34:76-83.
[本文引用: 1]

Tian Z J, Yi R, Chen J R, Guo Q Q, Zhang X W. Cloning and expression of cellulose synthase gene in ramie [Boehmeria nivea (Linn.) Gaud.]
Acta Agron Sin, 2008,34:76-83 (in Chinese with English abstract).
[本文引用: 1]

[5] 唐映红, 陈建荣, 刘芳, 袁有美, 郭清泉, 昌洪涛. 苎麻肉桂酰辅酶A还原酶基因cDNA序列的克隆与分析
作物学报, 2015,41:1324-1332.
[本文引用: 1]

Tang Y H, Chen J R, Liu F, Yuan Y M, Guo Q Q, Chang H T. cDNA cloning and analysis of cinnamoyl-CoA reductase gene from Boehmeria nivea
Acta Agron Sin, 2015,41:1324-1332 (in Chinese with English abstract).
[本文引用: 1]

[6] Liu F, Chen J, Tang Y, Chang H, Yuan Y, Guo Q. Isolation and characterization of cinnamate 4-hydroxylase gene from cultivated ramie (Boehmeria nivea)
Biotechnol Biotechnol Equip, 2018,32:324-331.
[本文引用: 1]

[7] Tang Y, Liu F, Mao K, Xing H, Chen J, Guo Q. Cloning and characterization of the key 4-coumarate CoA ligase genes in Boehmeria nivea
South Afr J Bot, 2018,116:123-130.
[本文引用: 1]

[8] Tang Y, Liu F, Xing H, Mao K, Chen G, Guo Q, Chen J. Correlation analysis of lignin accumulation and expression of key genes involved in lignin biosynthesis of ramie (Boehmeria nivea)
Genes, 2019,10:389.
[本文引用: 1]

[9] Liu T, Zhu S, Tang Q, Chen P, Yu Y, Tang S. De novo assembly and characterization of transcriptome using Illumina paired-end sequencing and identification of CesA gene in ramie (Boehmeria nivea L. Gaud.)
BMC Genomics, 2013,14:125.
DOI:10.1186/1471-2164-14-125URLPMID:23442184 [本文引用: 1]
BACKGROUND: Ramie fiber, extracted from vegetative organ stem bast, is one of the most important natural fibers. Understanding the molecular mechanisms of the vegetative growth of the ramie and the formation and development of bast fiber is essential for improving the yield and quality of the ramie fiber. However, only 418 expressed tag sequences (ESTs) of ramie deposited in public databases are far from sufficient to understand the molecular mechanisms. Thus, high-throughput transcriptome sequencing is essential to generate enormous ramie transcript sequences for the purpose of gene discovery, especially genes such as the cellulose synthase (CesA) gene. RESULTS: Using Illumina paired-end sequencing, about 53 million sequencing reads were generated. De novo assembly yielded 43,990 unigenes with an average length of 824 bp. By sequence similarity searching for known proteins, a total of 34,192 (77.7%) genes were annotated for their function. Out of these annotated unigenes, 16,050 and 13,042 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 19,846 unigenes were mapped to 126 KEGG pathways, and 565 genes were assigned to http://starch and sucrose metabolic pathway which was related with cellulose biosynthesis. Additionally, 51 CesA genes involved in cellulose biosynthesis were identified. Analysis of tissue-specific expression pattern of the 51 CesA genes revealed that there were 36 genes with a relatively high expression levels in the stem bark, which suggests that they are most likely responsible for the biosynthesis of bast fiber. CONCLUSION: To the best of our knowledge, this study is the first to characterize the ramie transcriptome and the substantial amount of transcripts obtained will accelerate the understanding of the ramie vegetative growth and development mechanism. Moreover, discovery of the 36 CesA genes with relatively high expression levels in the stem bark will present an opportunity to understand the ramie bast fiber formation and development mechanisms.

[10] Chen J, Pei Z, Dai L, Wang B, Liu L, An X, Peng D. Transcriptome profiling using pyrosequencing shows genes associated with bast fiber development in ramie (Boehmeria nivea L.)
BMC Genomics, 2014,15:919.
[本文引用: 5]

[11] Batista P J, Chang H Y. Long noncoding RNAs: cellular address codes in development and disease
Cell, 2013,152:1298-1307.
[本文引用: 1]

[12] Wang J, Huang J S, Hao X Y, Feng Y P, Cai Y J, Sun L Q. miRNAs expression profile in bast of ramie elongation phase and cell wall thickening and end wall dissolving phase
Mol Biol Rep, 2014,41:901-907.
[本文引用: 2]

[13] Li X, Yang L, Chen L L. The biogenesis, functions, and challenges of circular RNAs
Mol Cell, 2018,71:428-442.
[本文引用: 1]

[14] Zhang P, Fan Y, Sun X, Chen L, Terzaghi W, Bucher E. A large-scale circular RNA profiling reveals universal molecular mechanisms responsive to drought stress in maize and Arabidopsis
Plant J, 2019,98:697-713.
[本文引用: 2]

[15] Zuo J, Wang Q, Zhu B, Luo Y, Gao L. Deciphering the roles of circRNAs on chilling injury in tomato
Biochem Biophys Res Commun, 2016,479:132-138.
[本文引用: 2]

[16] Wang Y, Wang Q, Gao L, Zhu B, Luo Y, Deng Z, Zuo J. Integrative analysis of circRNAs acting as ceRNAs involved in ethylene pathway in tomato
Physiol Plant, 2017,16:311-321.
[本文引用: 2]

[17] Yin J, Liu M, Ma D, Wu J, Han B. Identification of circular RNAs and their targets during tomato fruit ripening
Postharvest Biol Technol, 2018,136:90-98.
[本文引用: 1]

[18] Wang Y, Yang M, Wei S, Qin F, Zhao H, Suo B. Identification of circular RNAs and their targets in leaves of Triticum aestivum L. under dehydration stress
Front Plant Sci, 2016,7:2024.
DOI:10.3389/fpls.2016.02024URLPMID:28105043 [本文引用: 2]
Circular RNAs (circRNAs) are a type of newly identified non-coding RNAs through high-throughput deep sequencing, which play important roles in miRNA function and transcriptional controlling in human, animals, and plants. To date, there is no report in wheat seedlings regarding the circRNAs identification and roles in the dehydration stress response. In present study, the total RNA was extracted from leaves of wheat seedlings under dehydration-stressed and well-watered conditions, respectively. Then, the circRNAs enriched library based deep sequencing was performed and the circRNAs were identified using bioinformatics tools. Around 88 circRNAs candidates were isolated in wheat seedlings leaves while 62 were differentially expressed in dehydration-stressed seedlings compared to well-watered control. Among the dehydration responsive circRNAs, six were found to act as 26 corresponding miRNAs sponges in wheat. Sixteen circRNAs including the 6 miRNAs sponges and other 10 randomly selected ones were further validated to be circular by real-time PCR assay, and 14 displayed consistent regulation patterns with the transcriptome sequencing results. After Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the targeted mRNAs functions, the circRNAs were predicted to be involved in dehydration responsive process, such as photosynthesis, porphyrin, and chlorophyll metabolism, oxidative phosphorylation, amino acid biosynthesis, and metabolism, as well as plant hormone signal transduction, involving auxin, brassinosteroid, and salicylic acid. Herein, we revealed a possible connection between the regulations of circRNAs with the expressions of functional genes in wheat leaves associated with dehydration resistance.

[19] Gao Z, Li J, Luo M, Li H, Chen Q, Wang L, Song S, Zhao L, Xu W, Zhang C. Characterization and cloning of grape circular RNAs identified the cold resistance-related Vv-circATS1
Plant Physiol, 2019,180:966-985.
DOI:10.1104/pp.18.01331URLPMID:30962290 [本文引用: 3]
Circular RNAs (circRNAs) are widely distributed and play essential roles in a series of developmental processes, although none have been identified or characterized in grapevines (Vitis vinifera). In this study, we characterized the function of grape circRNA and uncovered thousands of putative back-splicing sites by global transcriptome analysis. Our results indicated that several reported circRNA prediction algorithms should be used simultaneously to obtain comprehensive and reliable circRNA predictions in plants. Furthermore, the length of introns flanking grape circRNAs was closely related to exon circularization. Although the longer introns flanking grape circRNAs appeared to circularize more efficiently, a 20- to 50-nt region seemed large enough to drive grape circRNA biogenesis. In addition, the endogenous introns flanking circularized exon(s) in conjunction with reverse complementary sequences could support the accurate and efficient circularization of various exons in grape, which constitutes a new tool for exploring the functional consequences caused by circRNA expression. Finally, we identified 475 differentially expressed circRNAs in grape leaves under cold stress. Overexpression of Vv-circATS1, a circRNA derived from glycerol-3-P acyltransferase, improved cold tolerance in Arabidopsis (Arabidopsis thaliana), while the linear RNA derived from the same sequence cannot. These results indicate the functional difference between circRNA and linear RNA, and provide new insight into plant abiotic stress resistance.

[20] Luan M, Jian J, Chen P, Chen J, Chen J, Gao Q, Gao G, Zhou J H, Chen K, Guang X, Chen J, Zhang Q, Wang X, Fang L, Sun Z, Bai M, Fang X, Zhao S, Xiong H, Yu C, Zhu A. Draft genome sequence of ramie, Boehmeria nivea (L.) Gaudich
Mol Ecol Resour, 2018,18:639-645.
URLPMID:29423997 [本文引用: 1]

[21] Gao Y, Wang J, Zhao F. CIRI: an efficient and unbiased algorithm for de novo circular RNA identification
Genome Biol, 2015,16:4.
[本文引用: 1]

[22] Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, Maier L, Mackowiak S D, Gregersen L H, Munschauer M. Circular RNAs are a large class of animal RNAs with regulatory potency
Nature, 2013,495:333.
[本文引用: 1]

[23] Li L, Guo J, Chen Y, Chang C, Xu C. Comprehensive CircRNA expression profile and selection of key CircRNAs during priming phase of rat liver regeneration
BMC Genomics, 2017,18:80.
URLPMID:28086788 [本文引用: 1]

[24] Love M I, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2
Genome Biol, 2014,15:550.
URLPMID:25516281 [本文引用: 1]

[25] Ferreira J, Zwinderman A. On the Benjamini-Hochberg method
Ann Statist, 2006,34:1827-1849.
[本文引用: 1]

[26] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method
Methods, 2001,25:402-408.
[本文引用: 1]

[27] Zhong R, Ye Z. Secondary cell walls: biosynthesis, patterned deposition and transcriptional regulation
Plant Cell Physiol, 2015,56:195-214.
[本文引用: 1]

[28] Speicher T, Li P, Wallace I. Phosphor regulation of the plant cellulose synthase complex and cellulose synthase-like proteins
Plants, 2018,7:52.
[本文引用: 1]

[29] Gui J, Luo L, Zhong Y, Sun J, Umezawa T, Li L. Phosphorylation of LTF1, an MYB transcription factor in populus, acts as a sensory switch regulating lignin biosynthesis in wood cells
Mol Plant, 2019,12:1325-1337.
URLPMID:31145998 [本文引用: 1]

[30] Liu C, Yu H, Li L. SUMO modification of LBD30 by SIZ1 regulates secondary cell wall formation in Arabidopsis thaliana
PLoS Genet, 2019,15:e1007928.
[本文引用: 1]

[31] Huang J H, Qi Y P, Wen S X, Guo P, Chen X M, Chen L S. Illumina microRNA profiles reveal the involvement of miR397a in Citrus adaptation to long-term boron toxicity via modulating secondary cell-wall biosynthesis
Sci Rep, 2016,6:22900.
[本文引用: 1]

[32] Sun X, Wang C, Xiang N, Li X, Yang S, Du J, Yang Y, Yang Y. Activation of secondary cell wall biosynthesis by miR319- targeted TCP4 transcription factor
Plant Biotechnol J, 2017,15:1284-1294.
[本文引用: 1]

[33] 骆甲, 王型力, 孙志超, 吴迪, 张玮, 王正加. 植物环状RNA研究进展
遗传, 2018,40:467-477.
[本文引用: 1]

Luo J, Wang X L, Sun Z C, Wu D, Zhang W, Wang Z J. Progress in circular RNAs of plants
Hereditas, 2018,40:467-477 (in Chinese with English abstract).
[本文引用: 1]

[34] Chu Q J, Zhang X C, Zhu X T, Liu C, Mao L F, Ye C Y, Zhu Q H, Fan L J. PlantcircBase: a database for plant circular RNAs
Mol Plant, 2017,10:1126-1128.
DOI:10.1016/j.molp.2017.03.003URLPMID:28315753 [本文引用: 1]

[35] Wang K, Wang C, Guo B H, Song K, Shi C H, Jiang X, Wang K Y, Tan Y C, Wang L Q, Wang L, Li J J, Li Y, Cai Y, Zhao H W, Sun X Y. CropCircDB: a comprehensive circular RNA resource for crops in response to abiotic stress
Database, 2019. doi: 10.1093/database/baz053.
[本文引用: 1]

[36] Zhang X G, Ma X L, Ning L L, Li Z F, Zhao K K, Li K, He J L, Yin D M. Genome-wide identification of circular RNAs in peanut (Arachis hypogaea L.)
BMC Genomics, 2019,20:653.
[本文引用: 1]

[37] Zhang J, Liu R, Zhu Y, Gong J, Yin S, Sun P, Feng H, Wang Q, Zhao S J, Wang Z Y, Li G. Identification and characterization of circRNAs responsive to methyl jasmonate in Arabidopsis thaliana
Int J Mol Sci, 2020,21:792.
[本文引用: 2]

[38] Li E, Bhargava A, Qiang W, Friedmann M C, Forneris N, Savidge R A, Johnson L, Mansfield S, Ellis B, Douglas C. The Class II KNOX gene KNAT7 negatively regulates secondary wall formation in Arabidopsis and is functionally conserved in populus
New Phytol, 2012,194:102-115.
DOI:10.1111/j.1469-8137.2011.04016.xURLPMID:22236040 [本文引用: 1]
* The formation of secondary cell walls in cell types such as tracheary elements and fibers is a defining characteristic of vascular plants. The Arabidopsis transcription factor KNAT7 is a component of a transcription network that regulates secondary cell wall biosynthesis, but its function has remained unclear. * We conducted anatomical, biochemical and molecular phenotypic analyses of Arabidopsis knat7 loss-of-function alleles, KNAT7 over-expression lines and knat7 lines expressing poplar KNAT7. * KNAT7 was strongly expressed in concert with secondary wall formation in Arabidopsis and poplar. Arabidopsis knat7 loss-of-function alleles exhibited irregular xylem phenotypes, but also showed increased secondary cell wall thickness in fibers. Increased commitment to secondary cell wall biosynthesis was accompanied by increased lignin content and elevated expression of secondary cell wall biosynthetic genes. KNAT7 over-expression resulted in thinner interfascicular fiber cell walls. * Taken together with data demonstrating that KNAT7 is a transcriptional repressor, we hypothesize that KNAT7 is a negative regulator of secondary wall biosynthesis, and functions in a negative feedback loop that represses metabolically inappropriate commitment to secondary wall formation, thereby maintaining metabolic homeostasis. The conservation of the KNAT7 regulatory module in poplar suggests new ways to manipulate secondary cell wall deposition for improvement of bioenergy traits in this tree.

[39] Gong S, Huang G, Sun X, Qin L, Li Y, Zhou L, Li X. Cotton KNL1, encoding a class II KNOX transcription factor, is involved in regulation of fibre development
J Exp Bot, 2014,65:4133-4147.
URLPMID:24831118 [本文引用: 1]

[40] Li S, Chen M, Yu D, Ren S, Sun S, Liu L, Liu C M. EXO70A1-mediated vesicle trafficking is critical for tracheary element development in Arabidopsis
Plant Cell, 2013,25:1774-1786.
[本文引用: 1]

[41] Zhong R, Burk D H, Nairn C J, Wood-Jones A, Morrison W H, Ye Z H. Mutation of SAC1, an Arabidopsis SAC domain phosphoinositide phosphatase, causes alterations in cell morphogenesis, cell wall synthesis, and actin organization
Plant Cell, 2005,17:1449-1466.
[本文引用: 1]