棉花学报 ›› 2021, Vol. 33 ›› Issue (4): 319-327.doi: 10.11963/cs20200028
收稿日期:
2020-04-07
出版日期:
2021-07-15
发布日期:
2021-09-14
通讯作者:
喻树迅
E-mail:1721646511@qq.com;ysx195311@163.com
作者简介:
卞英杰(1993―),男,硕士研究生, 基金资助:
Bian Yingjie(),Wang Hantao,Wei Hengling,Zhang Meng,Li Yi,Yu Shuxun(
)
Received:
2020-04-07
Online:
2021-07-15
Published:
2021-09-14
Contact:
Yu Shuxun
E-mail:1721646511@qq.com;ysx195311@163.com
摘要:
【目的】 研究陆地棉DEAD-box RNA解旋酶基因GhRH39在叶片发育中的功能。【方法】 借助生物信息学方法分析GhRH39的基因结构和进化关系,利用实时定量聚合酶链反应PCR(Quantitative real-time polymerase chain reaction, qRT-PCR)分析该基因在棉花不同组织和叶片不同发育时期的表达情况,利用病毒诱导的基因沉默(Virus-induced gene silencing, VIGS)技术对该基因进行沉默。观察阳性植株表型,检测其光合色素含量变化,对阳性植株中叶绿体发育和光合色素合成相关基因进行表达量检测。【结果】 GhRH39编码620个氨基酸,且序列较为保守。qRT-PCR分析表明,该基因在根、茎、叶片、顶芽、花瓣、纤维中均有表达,在叶片中表达量较高,且在叶片中的表达量随着叶片发育进程而变化。利用VIGS技术成功降低了GhRH39的表达水平,基因沉默的阳性棉株出现失绿表型,叶绿素a、叶绿素b、类胡萝卜素3种光合色素含量均降低。阳性沉默棉株的叶绿体发育和光合色素合成相关基因表达量出现一定程度的下降。【结论】 GhRH39基因影响棉花叶绿素和类胡萝卜素合成,同时影响叶绿体发育过程。
卞英杰,王寒涛,魏恒玲,张蒙,李弈,喻树迅. 陆地棉叶片发育相关基因GhRH39克隆与功能分析[J]. 棉花学报, 2021, 33(4): 319-327.
Bian Yingjie,Wang Hantao,Wei Hengling,Zhang Meng,Li Yi,Yu Shuxun. Cloning and functional analysis of GhRH39, a gene related to leaf development, in upland cotton (Gossypium hirsutum L.)[J]. Cotton Science, 2021, 33(4): 319-327.
表 1
本研究中涉及的引物信息"
引物名称 Primer names | 引物序列 Primer sequences |
C-GhRH39-F | GGATCTTCCAGAGATATGATGCAGAAAGCTATGATGGC |
C-GhRH39-R | CTGCCGTTCGACGATTCAACTTGGTGTAAACGATTCTCTCT |
Q-Ghactin-F | ATCCTCCGTCTTGACCTTG |
Q-Ghactin-R | TGTCCGTCAGGCAACTCAT |
Q-GhRH39-F | GCAGAAAGGATAGAGGCGGCAA |
Q-GhRH39-R | TGCACAGATGCAGACACAGCTT |
TRV-GhRH39-F | AAGGTTACCGAATTCTCTAGAGCTGGACTTGGGTGTTAAT |
TRV-GhRH39-R | CGTGAGCTCGGTACCGGATCCTTTGAAGGTTTGGCTGGAG |
Q-CLPR3-F | CTTGCCTCCGTTGCCTCTTCTT |
Q-CLPR3-R | TCCACTTGAGCAGGAGTAGCCA |
Q-FZL-F | CTCAAGGGGGAATCGTCCACCA |
Q-FZL-R | TGTGTATCAGCAATGGCAGGACC |
Q-FTSH-F | AACTCCATTGGCGAGATCCGTG |
Q-FTSH-R | GACGTGCCTACTCCAACGAACA |
Q-SIGF-F | TTCATCATGAGCAAGCGGTCCC |
Q-SIGF-R | GCTGGCCAATGTTGTCAGATGC |
Q-HEMB1-F | TGCCTCGAACAATTCGTCTGCT |
Q-HEMB1-R | CTGGGACACCGCTTGCTTACAT |
Q-CHLI-F | TGAACAGGCTCAGAAGGTTGCC |
Q-CHLI-R | GTTGTGGACTTCCCAGTTCCCC |
Q-CHLG-F | ACAATCACCTGCGACTCCCAAC |
Q-CHLG-R | TAGTATCAGTGTCCGCCGCTCT |
Q-PDS-F | GGCTGTTGGGATCACCACACTT |
Q-PDS-R | CCACATCGATGCCGGCTAAGTT |
表 2
GhRH39启动子区顺式作用元件预测"
元件名称 Element | 位置 Locus of strand/bp | 序列 Sequences | 功能注释 Function annotation |
ARE | 1753(+)/1969(+) | AAACCA | 厌氧诱导Anaerobic induction |
Box4 | 375(+)/1563(-)/687(+)/496(+)/1281(-) | ATTAAT | 光响应Light responsiveness |
CGTCA-motif | 722(-) | CGTCA | 茉莉酸甲酯响应MeJA-responsiveness |
GA-motif | 1591(-) | ATAGATAA | 部分光响应元件 Part of a light responsive element |
GT1-motif | 199(+)/1315(-) | GGTTAA | 光响应Light responsiveness |
LAMP-element | 1401(-) | CTTTATCA | 部分光响应元件 Part of a light responsive element |
MBS | 546(+) | CAACTG | 干旱诱导的MYB结合位点MYB binding site involved in drought-inducibility |
O2-site | 125(+) | GATGATGTGG | 醇溶蛋白代谢调控 Zein metabolism regulation |
TC-rich repeats | 230(+)/1372(+) | GTTTTCTTAC | 防卫与胁迫响应 Defense and stress responsiveness |
WUN-motif | 1188(+) | AAATTTCCT | 损伤响应Wound responsiveness |
[1] | 吴吉祥, 朱军, 季道藩, 等. 陆地棉产量性状的遗传效应及其与环境互作的分析[J]. 遗传, 1995, 17(5):1-4. |
Wu Jixiang, Zhu Jun, Ji Daofan, et al. Analysis of genetic effect ×environment interactions for yield traits in upland cotton[J]. Hereditas, 1995, 17(5):1-4. | |
[2] |
Whatley J M. Variations in the basic pathway of choloplast development[J]. New Phytologist, 1977, 78(2):407-420.
doi: 10.1111/nph.1977.78.issue-2 |
[3] | Zybailov B, Rutschow H, Friso G, et al. Sorting signals, N-terminal modifications and abundance of the chloroplast proteome[J/OL]. PLoS One, 2008, 3(4): e1994[2020-04-01]. https://doi.org/10.1371/journal.pone.0001994. |
[4] |
Zhang T, Feng P, Li Y F, et al. VIRESCENT-ALBINO LEAF 1 regulates leaf colour development and cell division in rice[J]. Journal of Experimental Botany, 2018, 69(20):4791-4804.
doi: 10.1093/jxb/ery250 pmid: 30102358 |
[5] | 许瑞瑞, 高明刚, 李明, 等. 黄瓜RNA解旋酶基因家族的生物信息学鉴定和表达分析[J]. 上海交通大学学报 (农业科学版), 2018, 36(3):86-96. |
Xu Ruirui, Gao Minggang, Li Ming, et al. Bioinformatics and expression analysis of RNA helicase gene family in cucumber[J]. Journal of Shanghai Jiao Tong University (Agricultural Science), 2018, 36(3):86-96. | |
[6] |
Owttrim G W. RNA helicases and abiotic stress[J]. Nucleic Acids Research, 2006, 34(11):3220-3230.
pmid: 16790567 |
[7] |
Vashisht A A, Tuteja N. Stress responsive DEAD-box helicases: a new pathway to engineer plant stress tolerance[J]. Journal of Photochemistry and Photobiology B-Biology, 2006, 84(2):150-160.
pmid: 16624568 |
[8] |
He J N, Duan Y, Hua D P, et al. DEXH box RNA helicase-mediated mitochondrial reactive oxygen species production in Arabidopsis mediates crosstalk between abscisic acid and auxin signaling[J]. The Plant Cell, 2012, 24(5):1815-1833.
doi: 10.1105/tpc.112.098707 |
[9] |
Ghazala N, Hunseung K. Chloroplast- or mitochondria- targeted DEAD-Box RNA helicases play essential roles in organellar RNA metabolism and abiotic stress responses[J]. Frontiers in Plant Science, 2017, 8:871-881.
doi: 10.3389/fpls.2017.00871 pmid: 28596782 |
[10] |
Wang Y C, Duby G, Purnelle B, et al. Tobacco VDL gene encodes a plastid DEAD-box RNA helicase and is involved in chloroplast differentiation and plant morphogenesis[J]. The Plant Cell, 2000, 12(11):2129-2142.
doi: 10.1105/tpc.12.11.2129 |
[11] | 孔荣荣. 水稻DEAD-box RNA解旋酶蛋白基因TCD33功能研究[D]. 上海:上海师范大学, 2019. |
Kong Rongrong. Functional research of a DEAD-box RNA helicase gene TCD33 in rice (Oryza sativa L.)[D]. Shanghai: Shanghai Normal University, 2019. | |
[12] |
Nishimura K, Ashida H, Ogawa T, et al. A DEAD box protein is required for formation of a hidden break in Arabidopsis chloroplast 23S rRNA[J]. The Plant Journal, 2010, 63(5):766-777.
doi: 10.1111/j.1365-313X.2010.04276.x pmid: 20561259 |
[13] |
Lescot M. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J]. Nucleic Acids Research, 2002, 30(1):325-327.
doi: 10.1093/nar/30.1.325 |
[14] |
Sudhir K, Glen S, Koichiro T. MEGA7: Molecular evolutionary genetics analysis version7.0 for bigger datasets[J]. Molecular Biology and Evolution, 2016, 33(7):1870-1874.
doi: 10.1093/molbev/msw054 pmid: 27004904 |
[15] |
Schmittgen T D, Livak K J. Analyzing real-time PCR data by the comparative C(T) method[J]. Nature Protocols, 2008, 3(6):1101-1108.
pmid: 18546601 |
[16] |
Gu Z H, Huang C J, Li F F, et al. A versatile system for functional analysis of genes and microRNAs in cotton[J]. Plant Biotechnology Journal, 2014, 12(5):638-649.
doi: 10.1111/pbi.12169 |
[17] | Lichtenthaler H K. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes[J]. Methods in Enzymology, 1987, 148:350-382. |
[18] |
Sun J, Zheng T H, Yu J, et al. TSV, a putative plastidic oxidoreductase, protects rice chloroplasts from cold stress during development by interacting with plastidic thioredoxin Z[J]. New Phytologist, 2017, 215(1):240-255.
doi: 10.1111/nph.2017.215.issue-1 |
[19] |
Wu H T, Tian Y, Wan Q, et al. Genetics and evolution of MIXTA genes regulating cotton lint fiber development[J]. New Phytologist, 2017, 217(2):883-895.
doi: 10.1111/nph.14844 |
[20] | 许瑞瑞, 李高青, 刘彩云, 等. 低温胁迫对番茄RNA解旋酶SlDEAD34基因表达的影响[J]. 上海交通大学学报 (农业科学版), 2017, 3(35):76-81. |
Xu Ruirui, Li Gaoqing, Liu Caiyun, et al. Gene expression analysis of RNA helicase gene SIDEAD34 under low temperature stress in tomato[J]. Jouranl of Shanghai Jiao Tong University (Agricultural Science), 2017, 3(35):76-81. | |
[21] | 张建苓. 番茄DEAD-box解旋酶基因SlDEAH1的克隆与功能研究[D]. 重庆: 重庆大学, 2013. |
Zhang Jianling. Cloning and functional analysis of a DEAD-box helicase gene SlDEAH1 in tomato[D]. Chongqing: Chongqing University, 2013. | |
[22] | 齐盛东. 拟南芥RNA解旋酶AtHelps调控盐胁迫抗性的分子机理[D]. 泰安:山东农业大学, 2013. |
Qi Shengdong. Molecular dissection of Arabidopsis RNA helicase AtHelps in regulation of salt stress tolerance[D]. Tai’an: Shandong Agricultural University, 2013. | |
[23] |
Chen J, Zhang Y J, Liu J B, et al. Genome-wide analysis of the RNA helicase gene family in Gossypium raimondii[J]. International Journal of Molecular Sciences, 2014, 15(3):4635-4656.
doi: 10.3390/ijms15034635 pmid: 24642883 |
[24] |
He P, Wu S Y, Jiang Y L, et al. GhYGL1d, a pentatricopeptide repeat protein, is required for chloroplast development in cotton[J]. BMC Plant Biology, 2019, 19(1):350-361.
doi: 10.1186/s12870-019-1945-1 |
[25] |
Chi W, He B Y, Mao J, et al. The function of RH22, a DEAD RNA helicase, in the biogenesis of the 50S ribosomal subunits of Arabidopsis chloroplasts[J]. Plant Physiology, 2012, 158(2):693-707.
doi: 10.1104/pp.111.186775 |
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