棉花学报 ›› 2022, Vol. 34 ›› Issue (2): 79-92.doi: 10.11963/cs20210047
• 研究与进展 • 下一篇
收稿日期:
2021-07-06
出版日期:
2022-03-15
发布日期:
2022-07-19
通讯作者:
朱龙付
E-mail:lfzhu@mail.hzau.edu.cn
作者简介:
周雪慧(1994―),女,硕士研究生, 基金资助:
Zhou Xuehui(), Gao Erlin, Wang Yujing, Li Yanlong, Yuan Daojun, Zhu Longfu*(
)
Received:
2021-07-06
Online:
2022-03-15
Published:
2022-07-19
Contact:
Zhu Longfu
E-mail:lfzhu@mail.hzau.edu.cn
摘要:
【目的】 克隆抗病相关基因GhROP6,解析其作用机制,为开展棉花抗病分子育种提供理论基础。【方法】 利用生物信息学方法系统分析了陆地棉Rho鸟苷三磷酸酶基因(Rho-related guanosine triphosphatase from plants, ROP)家族成员在染色体上的分布及组织表达模式。克隆了GhROP6 (Gh_A01G1392.1)基因,通过实时荧光定量聚合酶链式反应、病毒诱导的基因沉默(virus-induced gene silencing, VIGS)技术、拟南芥遗传转化技术及代谢物测定等对该基因进行功能分析。【结果】 在陆地棉中鉴定到28个ROP,其编码的多肽均含有ROP结构,包括4个GTP/GDP结合域、与下游靶蛋白结合的效应结构域和C末端的可变区域。染色体定位分析发现陆地棉ROP家族中24个基因对称分布在A亚基因组和D亚基因组中,另有3个基因分布在D亚基因组。实时荧光定量聚合酶链式反应分析发现,GhROP6在棉花不同器官表达量不同,在花瓣、柱头、开花后10 d的纤维中表达量较高,并受茉莉酸甲酯诱导上调表达。抑制GhROP6表达会降低茉莉酸生物合成相关基因GhLOX1、GhOPR3-1、GhOPR3-3、GhAOC1、GhAOS和茉莉酸信号通路相关基因GhMYC2的表达水平,削弱木质素合成相关基因GhCCR-1、GhF5H-1、GhCCoAOMT-2和GhCCoAOMT-3的表达,从而降低棉花对黄萎病的抗性;超表达组成型激活的GhROP6能增加转基因拟南芥茉莉酸-异亮氨酸含量和木质素含量,增强其对黄萎病菌抗性。【结论】 GhROP6可能通过茉莉酸合成和信号通路以及木质素合成代谢参与棉花抗黄萎病反应。
周雪慧, 高二林, 王钰静, 李焱龙, 袁道军, 朱龙付. GhROP6通过调控茉莉酸合成与木质素代谢参与棉花抗黄萎病反应[J]. 棉花学报, 2022, 34(2): 79-92.
Zhou Xuehui, Gao Erlin, Wang Yujing, Li Yanlong, Yuan Daojun, Zhu Longfu. GhROP6 involved in cotton resistance to Verticillium wilt through regulating jasmonic acid synthesis and lignin metabolism[J]. Cotton Science, 2022, 34(2): 79-92.
附表1
本研究所用的引物序列"
引物名称Primer name | 序列Sequence | 用途Purpose |
---|---|---|
qGhROP6-F | GCTCATCTCCTACACCAGCAATAC | 检测相应基因的表达水平 detect gene expression by qRT-PCR |
qGhROP6-R | CAGCAGTATCCCACAATCCAAG | |
GhLOX1-F | TAGAGAGGACATTTTGCCCTGG | |
GhLOX1-R | GGTCAAGGTCGTCCAGAGATTTTA | |
GhAOS1-F | CGGATTAGAGCCTCAGTGTCGG | |
GhAOS1-R | ATCTTGAGAAATGAAAGGACCAGG | |
GhAOS2-F | TGCCACCTGGTCCTTTCATTTC | |
GhAOS2-R | GCGTGTTTGGGCTCGGAAGGGTCG | |
GhAOC1-F | CAACCCCTTCACTACCACTGCC | |
GhAOC1-R | AGGGCTGCTTCTGTCTCTCTCG | |
GhOPR3-1-F | ATGCTGTTCATGCCAAAGGAGG | |
GhOPR3-1-R | TTTCTGATGTTTCCAGGGGTCG | |
GhMYC2-F | GCTCCGCCACTACCGTGCTC | |
GhMYC2-R | CTCGAAGCACTTTTTTACGGTGTTC | |
GhCCR-1-F | ATTGTTATGGGAAGGCAGTGGC | |
GhCCR-1-R | ACGAGAAGGTGTGCTAATGCG | |
GhF5H-1-F | TTGGAGGCAGATGCGAAAGATT | |
GhF5H-1-R | TCCTCTTGCCCGTGTTTGTTAC | |
GhCCoAOMT-2-F | TACGACAACACGCTGTGGAATGGG | |
GhCCoAOMT-2-R | CATCGCCGACAGGAAACATACAGA | |
GhCCoAOMT-3-F | GAGACCAGTGTGTATCCGAGGG | |
GhCCoAOMT-3-R | CAAGGGCAGTGGCTAAGAGAGA | |
GhUB7-F | GAAGGCATTCCACCTGACCAAC | |
GhUB7-R | CTTGACCTTCTTCTTCTTGTGCTTG | |
AtPAL1-F | CTTGTCAGGAGCAACACCATCA | |
AtPAL1-R | AACGAGCAAGGCATATTTGAAGAG | |
AtPAL2-F | CTACCCCATCAACCTGAACCCA | |
AtPAL2-R | TATGGTCCAGAAGCGGATGTGT | |
AtC4H-F | GCTTAGCAACAATGGTGGAATG | |
AtC4H-R | CATCCTTGGTATTACTTTGGGTCG | |
AtF5H-F | TCCTCTTGCCCGTGTTTGTTAC | |
AtF5H-R | TTGGAGGCAGATGCGAAAGATT | |
AtAOS1-F | TTTTATCGCCGAGAATCCAC | |
AtAOS1-R | CCTCCGCTAATCGGTTATGA | |
AtAOC1-F | AACTGAGCGTGTACGAAATCAAT | |
AtAOC1-R | CAAACATACTGCATTCACAAGGA | |
AtOPR3-F | CGGCGTTGGCAGAGTATTAT | |
AtOPR3-R | GCGAGCTTTGAGCCATTAAC | |
AtMYC2-F | ACGACTGAAACAACTCCGACG | |
AtMYC2-R | AACCGTCGTATGATTTCTCCG | |
RT-GhROP6-F | TCCTACACCAGCAATACTTTC | RT-PCR检测GhROP6表达水平 Detect the expression of GhROP6 by RT-PCR |
RT-GhROP6-R | GGAGGAATTAAGAAAGCTGAT | |
VIGS-GhROP6-F | CAGTGCCCATTACCACAGCC | 构建GhROP6沉默载体Construction of GhROP6 silencing vector |
VIGS-GhROP6-R | AGGAAAGTGTGAGAACACAAAGGG | |
GhROP6-F | ATGAGTGCATCAAGGTTCATCA | 扩增 GhROP6 基因 Amplification of full-length GhROP6 gene |
GhROP6-R | TCACAATATCGAGCAGGCCTT | |
CA1-GhROP6-F | ACAATAATTGAAGCAAGAG | 构建组成型激活的GhROP6载体 Construction of constitutively active GhROP6 vector |
CA1-GhROP6-R | GTGACGTGTGCCGTCGGCAAGA | |
CA2-GhROP6-F | TCACTGTTGGTGACGGTGCA | |
CA2-GhROP6-R | ATTCCGGAGTTGAGTCATACTT | |
DN1-GhROP6-F | ATTACTTTCACTTTTGCAGCAT | 构建组成型失活的GhROP6载体Construction of dominant negative GhROP6 vector |
DN1-GhROP6-R | CGGCAAGAGAATGCATGCT | |
DN2-GhROP6-F | AAGGAGTGCATGCTCATCTC | |
DN2-GhROP6-R | ACCCTGGAGCAGTGCCCATT |
附表2
陆地棉中ROP基因基本信息"
基因ID Gene ID | 长度 length/bp | 染色体位置 Chromosome localization | 基因ID Gene ID | 长度 length/bp | 染色体位置 Chromosome localization |
---|---|---|---|---|---|
Gh_A01G1392.1 | 597 | A01 | Gh_D03G0072 | 591 | D03 |
Gh_A02G0857 | 588 | A02 | Gh_D05G0243 | 603 | D05 |
Gh_A05G0179 | 630 | A05 | Gh_D05G1765 | 588 | D05 |
Gh_A05G1588 | 588 | A05 | Gh_D05G2437 | 591 | D05 |
Gh_A06G0026 | 591 | A06 | Gh_D06G0618 | 714 | D06 |
Gh_A06G0551 | 630 | A06 | Gh_D06G1409 | 645 | D06 |
Gh_A06G2039 | 594 | A06 | Gh_D06G2288 | 591 | D06 |
Gh_A08G0520 | 639 | A08 | Gh_D08G0612 | 630 | D08 |
Gh_A08G1258 | 627 | A08 | Gh_D08G1547 | 627 | D08 |
Gh_A11G1595 | 594 | A11 | Gh_D10G0271 | 603 | D10 |
Gh_A12G0343 | 597 | A12 | Gh_D11G1753 | 564 | D11 |
Gh_A12G2499 | 636 | A12 | Gh_D12G0319 | 597 | D12 |
Gh_D01G1636 | 552 | D01 | Gh_D12G2627 | 666 | D12 |
Gh_D02G0984 | 588 | D02 | Gh_Sca006742G01 | 591 |
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