利用病毒诱导的基因沉默cDNA文库高通量筛选鉴定棉花功能基因

doi:10.3724/SP.J.1006.2022.14210

作物学报 ›› 2022, Vol. 48 ›› Issue (12): 2967-2977.doi: 10.3724/SP.J.1006.2022.14210

• 作物遗传育种·种质资源·分子遗传学 • 下一篇

利用病毒诱导的基因沉默cDNA文库高通量筛选鉴定棉花功能基因

梁曦彤¹^,²(), 高先原¹, 周琳¹, 穆春¹, 杜明伟¹, 李芳军¹(), 田晓莉¹(), 李召虎¹

¹植物生长调节剂教育部工程研究中心 / 中国农业大学农学院, 北京100193
²盐城师范学院, 江苏盐城 224007

收稿日期:2021-11-10 接受日期:2022-03-25 出版日期:2022-12-12 网络出版日期:2022-04-19
通讯作者: 李芳军,田晓莉
作者简介:E-mail: liangxt116@163.com
基金资助:
国家重点研发计划项目“大田经济作物逆境生理及调控”(2018YFD1000903)

High throughput identification of cotton gene via screening cotton cDNA library of virus induced gene silencing

LIANG Xi-Tong¹^,²(), GAO Xian-Yuan¹, ZHOU Lin¹, MU Chun¹, DU Ming-Wei¹, LI Fang-Jun¹(), TIAN Xiao-Li¹(), LI Zhao-Hu¹

¹Engineering Research Center of Plant Growth Regulator, Ministry of Education / College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China
²Yancheng Teachers University, Yancheng 224007, Jiangsu, China

Received:2021-11-10 Accepted:2022-03-25 Published:2022-12-12 Published online:2022-04-19
Contact: LI Fang-Jun,TIAN Xiao-Li
Supported by:
National Key Research and Development Program of China “Physiological Basis and Regulation of Stress Resistance of Cash Crops in Field”(2018YFD1000903)

摘要/Abstract

摘要：

为快速、高通量研究棉花功能基因, 本研究以陆地棉欣试17为材料, 棉花GhCLA1为标记基因, 利用棉花病毒诱导的基因沉默(virus induced gene silencing, VIGS) cDNA文库对幼苗生长和盐胁迫应答相关基因进行筛选和鉴定。VIGS转化7~14 d观察植株长势和盐胁迫应答表型, 共获得8个与幼苗生长相关以及4个与盐胁迫应答相关的基因。水培条件下, VIGS沉默GhANT17、GhSTP14、GhUSPA、GhFES1、GhS15-4、GhRBL8基因对植株地上部生长较对照植株有明显的抑制作用; VIGS沉默GhOIDO基因促进了植株生长; VIGS沉默GhRBCSC1基因抑制叶片中叶绿素的合成。盐胁迫处理条件下, 与对照植株相比VIGS沉默GhATCYP1、GhSAC52基因提高了植株的耐盐性, VIGS沉默GhPSBW、GhRBCSC2的植株对盐胁迫更敏感。本研究建立了高通量筛选棉花功能基因的技术体系, 为快速挖掘、研究棉花功能基因提供了可行技术手段。

关键词: 棉花, VIGS cDNA文库, 盐胁迫, 幼苗生长

Abstract:

To rapidly and efficiently explore functional genes in cotton, we have developed a functional genomic screen based on virus induced gene silencing (VIGS) assays to identify key players controlling cotton seedling growth and salt response with Gossypium hirsutum Xinshi 17 as plant materials and GhCLA1 as a visual marker gene. After 7-14 days of Agrobacterium- mediated transformation of cotton VIGS cDNA library, the phenotype of seedling growth and salt stress response were recorded. A total of eight genes related to seedling growth and four genes related to salt stress response were obtained. In hydroponic conditions, silencing of GhANT17, GhSTP14, GhUSPA, GhFES1, GhS15-4, and GhRBL8 significantly hindered shoot growth, while the silencing of GhOIDO promoted plant growth. GhRBCSC1-silenced plants had albino leaves. Under salt stress, silencing of GhATCYP1 and GhSAC52 improved salt tolerance. GhPSBW- and GhRBCSC2-silenced plants were more sensitive to salt stress compared with the control plants. Here, we established a technical system for high-throughput screening of functional genes in cotton, which provided a feasible tool for rapid mining and research of cotton functional genomic.

Key words: cotton, VIGS cDNA library, salt tolerant, seedling growth

梁曦彤, 高先原, 周琳, 穆春, 杜明伟, 李芳军, 田晓莉, 李召虎. 利用病毒诱导的基因沉默cDNA文库高通量筛选鉴定棉花功能基因[J]. 作物学报, 2022, 48(12): 2967-2977.

LIANG Xi-Tong, GAO Xian-Yuan, ZHOU Lin, MU Chun, DU Ming-Wei, LI Fang-Jun, TIAN Xiao-Li, LI Zhao-Hu. High throughput identification of cotton gene via screening cotton cDNA library of virus induced gene silencing[J]. Acta Agronomica Sinica, 2022, 48(12): 2967-2977.

图/表 11

表1

表2

VIGS cDNA文库筛选获得的候选基因注释"

序号No.	基因ID Gene ID	基因注释 Gene annotation
1	Gohir.D09G2158, Gohir.A09G213678	40S核糖体蛋白S15-4 40S ribosomal protein S15-4
2	Gohir.D05G2788, Gohir.A05G2771	60S核糖体蛋白L10 60S ribosomal protein L10-like
3	Gohir.A02G1627, Gohir.D03G0176	60S核糖体蛋白L8 60S ribosomal protein L8
4	Gohir.A11G2127, Gohir.D11G2105	富含ACT结构域的蛋白ACR10 ACT domain-containing protein ACR10-like
5	Gohir.D01G0567	酰基载体蛋白2 Acyl carrier protein 2
6	Gohir.D13G0291	邻氨基苯甲酸合成酶β亚基2 Anthranilate synthase beta subunit 2
7	Gohir.D12G0929, Gohir.A12G0833	ATP合酶亚单位O ATP synthase subunit O
8	Gohir.D09G0993, Gohir.A09G0999	钙调素 Calmodulin
9	Gohir.A13G0243	叶绿体叶绿素II A-B结合蛋白 Chloroplast chlorophyl II A-B binding protein
10	Gohir.A12G2050, Gohir.D12G2242	真核细胞翻译起始因子2的α亚基 Eukaryotic translation initiation factor 2 subunit alpha
11	Gohir.A11G0557	Hsp70核苷酸交换因子fes1 Hsp70 nucleotide exchange factor fes1
12	Gohir.D08G1902	白细胞花青素双加氧酶ANT17 Leucoanthocyanidin dioxygenase ANT17
13	Gohir.D01G1750	苹果酸脱氢酶 Malate dehydrogenase
14	Gohir.A01G2084	非共生血红蛋白2 Non-symbiotic hemoglobin 2
15	Gohir.D04G0066	寡糖基转移酶复合物亚单位OSTC Oligosaccharyltransferase complex subunit OSTC-like
16	Gohir.A13G2343	氧戊二酸/铁依赖性加氧酶 Oxoglutarate/iron-dependent oxygenase
17	Gohir.D01G1561, Gohir.A01G1642	肽基脯氨酰顺反异构酶 Peptidyl-prolyl cis-trans isomerase
18	Gohir.D07G1064, Gohir.A07G1022	肽基脯氨酰顺反异构酶CYP71 Peptidyl-prolyl cis-trans isomerase CYP71
19	Gohir.D12G0718, Gohir.A02G1550	肽基脯氨酰顺反异构酶FKBP13 Peptidyl-prolyl cis-trans isomerase FKBP13
20	Gohir.A02G0314	光系统I反应中心XI亚基 Photosystem I reaction centre subunit XI
21	Gohir.D04G0349, Gohir.A05G3807	光系统II氧释放增强蛋白2 Photosystem II oxygen-evolving enhancer protein 2
22	Gohir.A08G1884, Gohir.D08G2068	光系统II反应中心W蛋白 Photosystem II reaction center W protein
23	Gohir.D08G2068, Gohir.A11G1277	光系统II反应中心W蛋白 Photosystem II reaction center W protein
24	Gohir.D13G2157	2-酮戊二酸依赖性双加氧酶AOP1 Probable 2-oxoglutarate-dependent dioxygenase AOP1
25	Gohir.D05G3057	功能未知蛋白质DUF1749 Protein of unknown function DUF1749
26	Gohir.D11G0269 Gohir.A11G0277	蛋白酪氨酸磷酸酶线粒体1亚型1 Protein-tyrosine phosphatase mitochondrial 1-like isoform 1
27	Gohir.D02G1705	丙酮酸脱氢酶E1组分α-1亚基 Pyruvate dehydrogenase E1 component subunit alpha-1
28	Gohir.D11G1696, Gohir.A11G1632	核酮糖二磷酸羧化酶小链 Ribulose bisphosphate carboxylase small chain
29	Gohir.D11G1697	核酮糖二磷酸羧化酶小链 Ribulose bisphosphate carboxylase small chain
30	Gohir.A11G1633	核酮糖二磷酸羧化酶小链 Ribulose bisphosphate carboxylase small chain
31	Gohir.A03G0224	核酮糖二磷酸羧化酶小链1A Ribulose bisphosphate carboxylase small chain 1A
32	Gohir.D03G1463	核酮糖二磷酸羧化酶小链1A Ribulose bisphosphate carboxylase small chain 1A
33	Gohir.D07G1809	核酮糖二磷酸羧化酶小链X1转录本 Ribulose bisphosphate carboxylase small chain, transcript variant X1
34	Gohir.A11G1632	核酮糖-1,5-二磷酸羧化酶/加氧酶小亚基(rbcS2b) Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS2b)
35	Gohir.D07G1759	核酮糖二磷酸羧化酶小链 Ribulose-bisphosphate carboxylase small chain
36	Gohir.D13G1755	Sec非依赖型蛋白移位酶TATA Sec-independent protein translocase protein TATA
37	Gohir.D01G1645	SKP1蛋白1A SKP1-like protein 1A
38	Gohir.A06G0566, Gohir.D06G0550	糖转运蛋白14 STP14 Sugar transport protein 14 STP14
39	Gohir.D13G0715	编码未知功能蛋白 Uncharacterized
40	Gohir.D12G1716	编码未知功能蛋白 Uncharacterized
41	Gohir.A13G0543	编码未知功能蛋白 Uncharacterized
42	Gohir.A03G0386, Gohir.D03G1298	广谱应激蛋白A Universal stress protein A-like protein

表2

表3

图1

图2

图3

图4

表4

图5

图6

图7

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基因Gene	引物Primer (5°-3°)
GhACTIN9	F: GCCTTGGACTATGAGCAGGA; R: AAGAGATGGCTGGAAGAGGA
GhRBCSC1	F: GCCAACAACGACATCACTTC; R: ATCGGGCAGGTATGAGAGAG
GhANT17	F: ATGGTGACCTCAGTGCTACC; R: ACCTCTTTGTCCTCCGACTC
GhSTP14	F: TAGAAACAAAGGGCGTCG; R: CGAAAGATACAAGGGAACTGC
GhUSPA	F: CGGTGTAGCAGTGGATTACTC; R: AGACTCGTCGCTTTGATGG
GhFES1	F: CAGGGTTGATTTGGTCCAC; R: CAGATTCCTTGAGTTTAGCCG
GhS15-4	F: CGATTTGGATGCTCTCCTTG; R: ACGCAGGTGGGTTCTAACTG
GhRBL8	F: CCACCCTTTCCGTTACAAG; R: GGCAACACATTTCCAACC
GhOIDO1	F: CAGCCATTGAGTTCCGTTC; R: TTCTGCTTCGTCTCCACTG
GhATCYP1	F: AGGAGTGTTCCAGGATTGC; R: CGGGCTATCTTGTAACCAAAG
GhSAC52	F: ATTGGAGGCACATTGAGC; R: ACGAGAACAGAGTAAGATTGGC
GhPSBW	F: AAAGGACCCGTTGAAAGG; R: GGCAAGTGTAGAAGGAGTTGAC
GhRBCSC2	F: ACAGATACTCAACCCAACGG; R: TTCTTCGGTGGTCCCTTAC

利用病毒诱导的基因沉默cDNA文库高通量筛选鉴定棉花功能基因

High throughput identification of cotton gene via screening cotton cDNA library of virus induced gene silencing

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基因名 Gene name	基因ID Gene ID	基因注释 Gene annotation
GhATCYP1	Gohir.D01G1561, Gohir.A01G1642	陆地棉肽基脯氨酰顺反异构酶G. hirsutum peptidyl-prolyl cis-trans isomerase
GhSAC52	Gohir.D05G2788, Gohir.A05G2771	陆地棉60S核糖体蛋白L10 G. hirsutum 60S ribosomal protein L10
GhPSBW	Gohir.D08G2068, Gohir.A11G1277	陆地棉光系统II反应中心W蛋白G. hirsutum photosystem II reaction center W protein
GhRBCSC2	Gohir.D11G1696, Gohir.A11G1632	核酮糖二磷酸羧化酶小链 Ribulose bisphosphate carboxylase small chain

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