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作物学报 ›› 2022, Vol. 48 ›› Issue (1): 63-75.doi: 10.3724/SP.J.1006.2022.01100

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦Ta4CL1基因的克隆及其在促进转基因拟南芥生长和木质素沉积中的功能

孟颖1(), 邢蕾蕾1, 曹晓红1, 郭光艳1, 柴建芳2,*(), 秘彩莉1,*()   

  1. 1河北师范大学生命科学学院, 河北石家庄 050024
    2河北省农林科学院遗传生理研究所 / 河北省植物转基因中心, 河北石家庄 050051
  • 收稿日期:2020-12-23 接受日期:2021-04-14 出版日期:2022-01-12 网络出版日期:2021-06-02
  • 通讯作者: 柴建芳,秘彩莉
  • 作者简介:E-mail: mengying941027@tom.com
  • 基金资助:
    河北省自然科学基金项目(C2019205102);河北省重点研发计划项目(20326348D);河北师范大学重点基金项目(L2019Z05);河北师范大学博士基金项目资助(L2018B14)

Cloning of Ta4CL1 and its function in promoting plant growth and lignin deposition in transgenic Arabidopsis plants

MENG Ying1(), XING Lei-Lei1, CAO Xiao-Hong1, GUO Guang-Yan1, CHAI Jian-Fang2,*(), BEI Cai-Li1,*()   

  1. 1Life Science College, Hebei Normal University, Shijiazhuang 050024, Hebei, China
    2Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forest Sciences, Plant Genetic Engineering Center of Hebei Province, Shijiazhuang 050051, Hebei, China
  • Received:2020-12-23 Accepted:2021-04-14 Published:2022-01-12 Published online:2021-06-02
  • Contact: CHAI Jian-Fang,BEI Cai-Li
  • Supported by:
    Natural Science Foundation of Hebei Province(C2019205102);Key Research and Development Projects of Hebei Province(20326348D);Key Foundation of Hebei Normal University(L2019Z05);Doctoral Foundation of Hebei Normal University(L2018B14)

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摘要：

4-香豆酸辅酶A连接酶(4CL; EC 6.2.1.12)位于苯丙烷途径分支点的上游, 是苯丙烷代谢途径的核心酶, 可产生木质素、黄酮类等化合物, 这些化合物对植物的生长发育及环境适应性均具有重要作用。在双子叶植物中, 有关4CL的研究较多, 而在单子叶植物尤其是作物中的研究相对较少。本研究利用RACE技术从普通小麦中克隆了一个4CL基因Ta4CL1。系统发育分析表明, Ta4CL1与水稻、玉米和高粱等植物中在木质素合成中具有重要作用的4CLs聚成一类; 利用Ta4CL1过表达、拟南芥4CLs突变体at4cl1、at4cl3和at4cl14cl3及其功能回复株系进行的分析表明, Ta4CL1与At4CL1功能相似, 在植物木质素合成中具有重要作用, 但未参与黄酮类化合物生物合成的调控过程; Ta4CL1是转基因拟南芥中4CL酶活性的主要贡献者。过表达Ta4CL1的转基因拟南芥叶片增大、茎更粗; Ta4CL1的表达还受茉莉酸甲酯(Methyl jasmonic acid, MeJA)、赤霉素(Gibberellin, GA)和生长素(Indoleacetic acid, IAA)等激素处理的影响。本研究为利用基因工程将Ta4CL1应用于改善小麦秸秆的利用效率提供了理论依据。

关键词: 4-香豆酸辅酶A连接酶, Ta4CL1, 木质素沉积, 促进生长, 小麦, 拟南芥

Abstract:

4-Coumarate:coenzyme A ligase (4CL; EC 6.2.1.12) acts upstream of the branch point of phenylpropanoid, which is a key enzyme in the phenylpropanoid metabolic pathways for monolignol and flavonoid biosynthesis, and these compounds play important roles in plant growth and development as well as stress adaptability. Many 4CLs had been extensively studied in dicotyledons, but their function in monocotyledons, especially in crops, was relatively poorly understood. In this study, Ta4CL1, which encoded 4-Coumarate:coenzyme A ligase, was isolated from common wheat by RACE technique. Polygenetic analysis revealed that Ta4CL1 could be clustered to the same group of 4CLs from rice, maize, and sorghum, which was mainly involved in lignin biosynthesis. Ta4CL1-overexpressed Arabidopsis lines, at4cl1, at4cl3, and at4cl14cl3 as well as their corresponding functional recovery lines were used to elucidate the function of Ta4CL1 in the phenylpropanoid metabolic pathway. The results suggested that Ta4CL1 had similar function with At4CL1 in regulating lignin biosynthesis but it had no effect on flavonoid biosynthesis. Ta4CL was the major contributor of 4CL enzyme activity in transgenic Arabidopsis plants. Overexpression of Ta4CL led to enlarged leaves and thickened stems in transgenic Arabidopsis seedlings, the expression of Ta4CL was also affected by MeJA, GA, and IAA treatments. These results provide the theoretical basis for improving the utilizing efficiency of plant straws using Ta4CL1 by genetic engineering.

Key words: 4-Coumaric coenzyme A ligase, Ta4CL1, lignin deposition, promote growth, wheat, Arabidopsis thaliana L.

表1

本研究的引物及序列"

引物Primer 序列Sequence (5′-3′) 用途Usage
Ta4CL1 F1 CCGCGGGGAGCAGATCATGAAAGGTTAC 3′RACE第1轮PCR 1st run PCR of 3′RACE
Ta4CL1 F2 ACATCAAGAAATTCGTCGCAAAGGAGGTT 3′RACE第2轮PCR 2nd run PCR of 3′RACE
Ta4CL1 R1/R2 AAGCAGTGGTATCAACGCAGAGTAC(T)30N-1N (N=A, C, G or T; N-1=A, G or C) 3′RACE
Ta4CL1 F3/F4 (T25)N-1N (N=A, C, G or T; N-1=A, G or C) 5′RACE
Ta4CL1 R3 AACCTCCTTTGCGACGAATTTCTTGATGT 5′RACE第1轮PCR 1st run PCR of 5′RACE
Ta4CL1 R4 F: TGTCTCCGGTGTGCAGCCATCCAT 5′RACE第2轮PCR 2nd run PCR of 5′RACE
Ta4CL1 F5/R5 F: CGCACGCACGCACACGCACAA
R: ATCAACATTACACAAGCAGGAAGAACCA		 全长cDNA克隆
Isolation of full-length cDNA
Ta4CL1 F6/R6 F: TCTAGAATGGGGTCTGTGCCGGAG (Xba I)
R: ggTACCCTAGCTTGGGATGCCGGC (Kpn I)		 Ta4CL1过表达载体构建
Construction of Ta4CL1-overexpression vector
Ta4CL1pro F1/R1 F:CCAGTGCCCTCCATCTCT
R:CGCCGCCGCAACCGACTC		 克隆Ta4CL1启动子
Cloning of Ta4CL1 promoter
Ta4CL1pro F2/R3 F: AAGCTTCCAGTGCCCTCCATCTCT (Hind III)
R: GAATTCTTCAACGATCGTGGTAGA (EcoR I)		 Ta4CL1pro:Gus载体构建
Construction of Ta4CL1 pro:Gus vector
GUS F: GGGCAGGCCAGCGTATCG
R: GTCCCGCTAGTGCCTTGTC		 鉴定Ta4CL1pro:Gus载体
Identification of Ta4CL1pro:Gus vector
Actin2
(At3g18780)		 F: GCTGAGAGATTCAGATGCCC
R: CTCGGCCTTGGAGATCCACA		 RT-PCR
Actin2
(At3g18780)		 F: TCGCTGACCGTATGAGCAAAG
R: TGTGAACGATTCCTGGACCTG		 qRT-PCR
AtC3H
(At2g40890)		 F: GTTGGACTTGACCGGATCTT
R: ATTAGAGGCGTTGGAGGATG		 qRT-PCR
AtCOMT1-1
(At5g54160)		 F: TTCCATTGCTGCTCTTTGTC
R: CATGGTGATTGTGGAATGGT		 qRT-PCR
AtCCoAOMT1
(At4g34050)		 F: CTCAGGGAAGTGACAGCAAA
R: GTGGCGAGAAGAGAGTAGCC		 qRT-PCR
AtCAD5
(At4g34230)		 F: TTGGCTGATTCGTTGGATTA
R: ATCACTTTCCTCCCAAGCAT		 qRT-PCR
AtF5H
(At4g36220)		 F: CTTCAACGTAGCGGATTTCA
R: AGATCATTACGGGCCTTCAC		 qRT-PCR
AtHCT
(At5g48930)		 F: GCCTGCACCAAGTATGAAGA
R: GACAGTGTTCCCATCCTCCT		 qRT-PCR
AtCCR1
(At1g15950)		 F: GTGCAAAGCAGATCTTCAGG
R: GCCGCAGCATTAATTACAAA		 qRT-PCR
Ta4CL1 F7/R7 F: TCGTCGCAAAGGAGGTTGTT
R: GCGGCCGAGTCTGGCTCT		 qRT-PCR
TaEF 1a
(M90077)		 F: CAGATTGGCAACGGCTACG
R: CGGACAGCAAAACGACCAAG		 qRT-PCR

图1

利用RACE技术克隆Ta4CL1全长cDNA 扩增Ta4CL1的5′ RACE (A)和3′ RACE (B)片段。1: 第1轮PCR; 2: 第2轮PCR; C: Ta4CL1的全长cDNA的扩增。1: Ta4CL1的全长cDNA。M: marker; 箭头所示为目的条带。"

图2

Ta4CL1及其同源蛋白的系统发育分析 利用各物种4CL的氨基酸序列构建的无根树(步长值: 1000次重复); 标尺0.05对应于0.05个氨基酸替换/位置。"

图3

利用Ta4CL1pro:Gus转基因拟南芥研究Ta4CL1的组织表达特性 A: 3周大的Ta4CL1pro:Gus转基因拟南芥幼苗的叶; B: A图中方框内叶片的放大图; C: 2周大的Ta4CL1pro:Gus转基因拟南芥幼苗的根; D: Ta4CL1pro:Gus转基因拟南芥主茎(6周)的横切面。A~C图中的标尺表示1 mm; D图中的标尺为0.18 mm。"

图4

GA、IAA和MeJA处理对Ta4CL1表达的影响"

图5

异源表达Ta4CL1的拟南芥纯合株系RNA水平的鉴定"

图6

异源表达Ta4CL1的拟南芥株系的表型观察 A: 正常生长2周的拟南芥幼苗; B: 正常生长6周的拟南芥幼苗; C: 各拟南芥株系的株高统计; D: 各拟南芥株系的直径统计; one-way ANOVA分析(字母不同表示差异显著, 含有相同字母表示差异不显著; P < 0.05)。Col-0: 野生型拟南芥; OX-1、-2和-3: 过表达Ta4CL1拟南芥纯合株系; Ta4CL1/at4cl1 L3、L9和L11: 在at4cl背景中过表达Ta4CL1的3个纯合株系; Ta4CL1/at4cl3 L10、L12和L13: 在at4cl3背景中过表达Ta4CL1的3个纯合株系; Ta4CL1/at4cl1 4cl3 L6、L10和L11: 在at4cl1 4cl3背景中过表达Ta4CL1的3个纯合株系。"

图7

异源表达Ta4CL1拟南芥株系中木质素含量测定 6周龄(A)和8周龄(B)拟南芥株系茎中木质素含量测定(P < 0.05)。"

图8

异源表达Ta4CL1拟南芥株系中类黄酮和花青素含量测定 正常生长1个月的拟南芥株系的叶片中类黄酮(A)和花青素(B)含量(P < 0.05)。"

图9

异源表达Ta4CL1拟南芥株系中4CL酶活性测定 正常生长4周的拟南芥株系莲座叶(A)和茎(B)中4CL酶活性的测定(P < 0.05)。"

图10

Ta4CL1过表达拟南芥株系中木质素合成途径4CL下游基因的表达分析 Col-0: 野生型拟南芥; OX-1, OX-2, OX-3: Ta4CL1过表达拟南芥株系。Student’s t测验, *P < 0.05, **P < 0.01, ***P < 0.001。"

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