VIGS技术在棉花全生长周期中的应用拓展研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0729

摘要/Abstract

摘要：

目的突破棉花VIGS技术仅应用于棉花子叶期的时间限制瓶颈，弥补棉花生殖生长时期VIGS技术的应用缺陷。方法以农大棉8号为材料，以GhCLA1为验证基因，对4日龄棉苗（子叶未展开）进行茎注射，对7日龄棉苗（子叶完全展开）进行子叶注射，对开花期和结铃期的棉花植株分别在茎节间、新生果枝基部、新生叶片等组织部位进行VIGS注射，利用表型观察和RT-PCR技术验证目的基因的沉默效率和持续时间。结果 4日龄棉花幼苗经茎VIGS注射后叶片白化率为80%，7日龄棉花幼苗经子叶VIGS注射后叶片白化率为100%，开花期和结铃期的棉花植株经新生果枝基部VIGS注射后白化率分别为90%和68.75％。结论获得了稳定的棉花全生长周期的VIGS应用技术体系。

关键词: 基因沉默, 棉花, 子叶, 茎, 生长期

Abstract:

Objective To break through the time limit bottleneck that cotton VIGS technology can only be applied in cotton cotyledon stage, and make up for the application deficiency of VIGS technology in cotton reproductive growth stage. Method Using Nongdamian 8 as the material and GhCLA1 as the verification gene, 4-day-old cotton seedlings' stem (cotyledon folded) were injected, 7-day-old cotton seedlings cotyledon (cotyledon fully unfolded) were injected, also flowering and bolling stages cotton stem and base of new fruit branches and new leaves were injected with VIGS respectively. Phenotype observation and RT-PCR were used to verify the silencing efficiency of target genes and photobleaching duration. Result The leaf photobleaching rate of 4-day-old cotton seedlings was 80% by VIGS injected to stems, that of 7-day-old cotton seedlings was 100% by VIGS injected to cotyledon, and that of flowering stage and bolling stage cotton was 90% and 68.75% by VIGS injection at the base of new fruit branches, respectively. Conclusion A stable VIGS application technology system for the whole growth cycle of cotton is obtained.

Key words: VIGS, cotton, cotyledon, stem, growing period

丁若羲, 豆硕, 安叶芝, 孔文慧, 郭文静, 张冬梅, 王省芬, 马峙英, 吴立柱. VIGS技术在棉花全生长周期中的应用拓展研究[J]. 生物技术通报, 2025, 41(2): 58-64.

DING Ruo-xi, DOU Shuo, AN Ye-zhi, KONG Wen-hui, GUO Wen-jing, ZHANG Dong-mei, WANG Xing-fen, MA Zhi-ying, WU Li-zhu. Extended Research on the Application of VIGS Technology in the Whole Growth Cycle of Cotton[J]. Biotechnology Bulletin, 2025, 41(2): 58-64.

图/表 6

表1 棉花不同组织位点进行VIGS处理后叶片白化率统计

Table 1 Statistics of leaf photobleaching rates after VIGS treatment at different tissue sites of cotton

注射部位和时期 Injection site and period		注射株数 Number of injected cotton seedlings	白化株数 Number of photobleaching cotton seedlings	白化率 Photobleaching rate/%	操作难易程度 Degree of difficulty for operation
4日龄幼苗茎注射		50	40	80.0±0.1	较难
7日龄幼苗子叶注射		45	45	100±0.1	容易
新生果枝基部（茎）注射	开花期	30	27	90.0±0.0	较难
新生果枝基部（茎）注射	结铃期	32	22	68.8±0.1	较难
节间注射	开花期	40	16	40.0±0.1	难
节间注射	结铃期	31	12	38.7±0.1	难
叶片注射	开花期	30	0	0±0.0	无法注入
叶片注射	结铃期	31	0	0±0.0	无法注入

图1 棉花幼苗VIGS处理后的白化表型A：未注射棉株； B：4日龄茎注射10 d后棉株；C：7日龄子叶注射10 d后棉株；标尺为2 cm

Fig. 1 Photobleaching phenotype of cotton seedling after VIGS treatmentA: Uninfected cotton seedling. B: Cotton plants of 10 d after stem injection at 4 d of age. C: Cotton plants of 10 d after cotyledon injection at 7 d of age. Bar=2 cm

图2 棉花成苗VIGS处理后的白化表型A：棉花成苗VIGS茎注射3周后表型观察；B：棉花成苗VIGS茎注射6周后表型观察；C：白化新叶与老叶对比；D：白化下行；E：茎白绿共存现象；标尺为10 cm

Fig. 2 Photobleaching phenotype of cotton plants after VIGS treatmentA: Phenotype of cotton plants at 3 weeks after VIGS injected to the cotton stem. B: Phenotype of cotton at 6 weeks after VIGS injected to cotton stem. C: The contrast between photobleaching leaves and old green leaves. D: Photobleaching downward. E: Coexistence of photobleaching and green on stem. Bar=10 cm

图3 棉铃白化表型观察A：易脱落的新生白化棉铃；B：未脱落的白化棉铃；C：成熟的白化棉铃；D：白化棉铃和正常棉铃的对比；标尺为2 cm

Fig. 3 Photobleaching phenotype of cotton bollA: Caducous photobleaching cotton bolls. B: Un-withered photobleaching cotton bolls. C: Mature photobleaching cotton bolls. D: The comparation of photobleaching cotton bolls and normal bolls. Bar=2 cm

图4 白化持续时长表型观察A：注射后3周的棉花表型；B：注射后6周的棉花表型；C：注射后12周的棉花表型；D：注射后19周的棉花表型；E：注射后22周的棉花表型；标尺为10 cm

Fig. 4 Phenotype observation of photobleaching durationA: Cotton phenotype at 3 weeks after injection. B: Cotton phenotype at 6 weeks after injection. C: Cotton phenotype at 12 weeks after injection. D: Cotton phenotype at 19 weeks after infection. E: Cotton phenotype at 22 weeks after infection. Bar=10 cm

图5 棉花GhCLA1基因沉默的鉴定A：棉花总RNA提取的电泳结果；B：棉花GhCLA1基因沉默的RT-RCR鉴定结果；C：棉花GhCLA1基因沉默的RT-qRCR鉴定结果，大写字母表示差异达到极显著水平（P<0.01）；M：2 000 bp marker；1：未经VIGS处理的幼苗真叶叶片；2：幼苗茎VIGS处理10 d后完全白化叶片；3：幼苗茎VIGS处理10 d后白绿共存叶片；4：未经VIGS处理的成苗正常叶片；5：成苗茎VIGS处理2个月后完全白化叶片；6：成苗茎VIGS处理2个月后完全白化的茎；7：成苗茎VIGS处理2个月后白绿共存茎；8：空白对照

Fig. 5 Identification of the GhCLA1 gene silence in cottonA: The electrophoresis result of cotton total RNA. B: The GhCLA1 gene silenced identification results by RT-RCR. C: The GhCLA1 gene silenced identification results by RT-qRCR. The capital letters indicate that the differences reached the highly significant level (P<0.01). M: 2 000 bp marker. 1: True leaf of cotton seedling untreated by VIGS. 2: The true photobleaching leaf at 10 d after VIGS treatment to seedling stems. 3: Coexisting photobleaching and green leaf at 10 d after VIGS treatment to seedling stems. 4: Normal leaf of cotton plant untreated by VIGS. 5: Completely photobleaching leaf at 2 months after VIGS treatment of plant stems. 6: Completely photobleaching stem at 2 months after VIGS treatment of plant stems. 7: Stems while coexisting of photobleaching and green leaves at 2 months after VIGS treatment of plant stems. 8: Blank control

参考文献 30

1	古丽斯坦·赛米, 刘隋赟昊, 郑蓓, 等. 棉花Gols基因家族的鉴定及其在干旱胁迫应答中的功能研究 [J]. 农业生物技术学报, 2023, 31(4): 704-717.
	GuLiSiTan SM, Liu SYH, Zheng B, et al. Identification of Gols gene family in cotton(Gossypium spp.) and its function analysis in drought stress response [J]. J Agric Biotechnol, 2023, 31(4): 704-717.
2	Paterson AH, Wendel JF, Gundlach H, et al. Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres [J]. Nature, 2012, 492(7429): 423-427.
3	Li FG, Fan GY, Wang KB, et al. Genome sequence of the cultivated cotton Gossypium arboreum [J]. Nat Genet, 2014, 46: 567-572.
4	唐丽媛, 蔡肖, 王海涛, 等. 棉花FLA基因家族的全基因组鉴定及GhFLA05在棉纤维发育中的功能分析 [J]. 中国农业科学, 2023, 56(23): 4602-4620.
	Tang LY, Cai X, Wang HT, et al. Genome-wide identification of cotton FLA gene family and functional analysis of GhFLA05 in cotton fiber development [J]. Sci Agric Sin, 2023, 56(23): 4602-4620.
5	高升旗, 邵武奎, 赵准, 等. 类钙调磷酸酶B亚基蛋白GhCBL3-A01调控棉花黄萎病抗性的功能分析 [J]. 棉花学报, 2023, 35(6): 447-458.
	Gao SQ, Shao WK, Zhao Z, et al. Functional analysis of cotton calcineurin B-like protein GhCBL3-A01 in regulating the resistance to Verticillium wilt [J]. Cotton Sci, 2023, 35(6): 447-458.
6	Scofield SR, Huang L, Brandt AS, et al. Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway [J]. Plant Physiol, 2005, 138(4): 2165-2173.
7	Gould B, Kramer EM. Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (Columbine, Ranunculaceae) [J]. Plant Methods, 2007, 3: 6.
8	Peng RH, Jones DC, Liu F, et al. From sequencing to genome editing for cotton improvement [J]. Trends Biotechnol, 2021, 39(3): 221-224.
9	Tian Y, Fang Y, Zhang KX, et al. Applications of virus-induced gene silencing in cotton [J]. Plants (Basel), 2024, 13(2): 272.
10	张晓红, 许佩阳, 闫绪, 等. 棉花GhFUL1基因及其启动子的克隆和功能分析 [J]. 华北农学报, 2023, 38(5): 60-68.
	Zhang XH, Xu PY, Yan X, et al. Cloning and functional analysis of GhFUL1 gene and its promoter in cotton [J]. Acta Agric Boreali Sin, 2023, 38(5): 60-68.
11	于晓红, 朱勇清, 林芝萍, 等. 亚洲棉GAE6-3A上游序列的分离及其在烟草中的表达 [J]. 植物生理学报, 2000, 26(2): 143-147, 180.
	Yu XH, Zhu YQ, Lin ZP, et al. Isolation of GAE6-3A5'-upstream fragment from Gossypium arboreum and its expression in tobacco [J]. Acta Photophysiol Sin, 2000, 26(2): 143-147, 180.
12	Ratcliff F, Martin-Hernandez AM, Baulcombe DC. Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing [J]. Plant J, 2001, 25(2): 237-245.
13	Bekele D, Tesfaye K, Fikre A. Applications of virus induced gene silencing (VIGS) in plant functional genomics studies [J]. J Plant Biochem Physiol, 2019, 7(1): 1-7.
14	高鹏飞, 席飞虎, 张泽宇, 等. 植物VIGS技术及其在林业科学中的研究进展 [J]. 生物技术通报, 2021, 37(5): 141-153.
	Gao PF, Xi FH, Zhang ZY, et al. Research progress of plant VIGS technology and its application in forestry science [J]. Biotechnol Bull, 2021, 37(5): 141-153.
15	李文辰, 刘鑫, 康越, 等. TRV病毒诱导大豆基因沉默体系优化及应用 [J]. 生物技术通报, 2023, 39(7): 143-150.
	Li WC, Liu X, Kang Y, et al. Optimization and application of tobacco rattle virus-induced gene silencing system in soybean [J]. Biotechnol Bull, 2023, 39(7): 143-150.
16	Zhang JX, Wang FR, Zhang CY, et al. A novel VIGS method by agroinoculation of cotton seeds and application for elucidating functions of GhBI-1 in salt-stress response [J]. Plant Cell Rep, 2018, 37(8): 1091-1100.
17	Gao X, Britt RC, Shan L, et al. Agrobacterium-mediated virus-induced gene silencing assay in cotton [J]. J Vis Exp, 2011(54): 2938.
18	Liu YL, Schiff M, Dinesh-Kumar SP. Virus-induced gene silencing in tomato [J]. Plant J, 2002, 31(6): 777-786.
19	Zhang J, Yu DS, Zhang Y, et al. Vacuum and co-cultivation agroinfiltration of (germinated) seeds results in tobacco rattle virus (TRV) mediated whole-plant virus-induced gene silencing (VIGS) in wheat and maize [J]. Front Plant Sci, 2017, 8: 393.
20	Ryu CM, Anand A, Kang L, et al. Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species [J]. Plant J, 2004, 40(2): 322-331.
21	Tuttle JR, Idris AM, Brown JK, et al. Geminivirus-mediated gene silencing from Cotton leaf crumple virus is enhanced by low temperature in cotton [J]. Plant Physiol, 2008, 148(1): 41-50.
22	Gao XQ, Wheeler T, Li ZH, et al. Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt [J]. Plant J, 2011, 66(2): 293-305.
23	Si ZF, Wu HT, Tian Y, et al. Visible gland constantly traces virus-induced gene silencing in cotton [J]. Front Plant Sci, 2022, 13: 1020841.
24	Zhao LJ, Wang YB, Cui RF, et al. Analysis of the histidine kinase gene family and the role of GhHK8 in response to drought tolerance in cotton [J]. Physiol Plant, 2023, 175(5): e14022.
25	张景霞, 王芙蓉, 高阳, 等. VIGS技术及其在棉花功能基因组研究中的应用进展 [J]. 棉花学报, 2015, 27(5): 469-473.
	Zhang JX, Wang FR, Gao Y, et al. Application of VIGS in studies of gene function in cotton [J]. Cotton Sci, 2015, 27(5): 469-473.
26	侯雪, 王姣姣, 张雯雯, 等. 辣椒乙烯反应抑制因子Cacl-6468的克隆及其抗根结线虫作用分析 [J]. 园艺学报, 2024, 51(4): 761-772.
	Hou X, Wang JJ, Zhang WW, et al. Cloning of pepper ethylene-responsive proteinase inhibitor Cacl-6468 and its effect on resistance to Meloidogyne incognita [J]. Acta Hortic Sin, 2024, 51(4): 761-772.
27	刘晓彬, 刘娜, 李福宽, 等. TRV介导的大豆基因瞬时沉默体系的建立 [J]. 中国农业科学, 2015, 48(12): 2479-2486.
	Liu XB, Liu N, Li FK, et al. Establishment of TRV-mediated transient gene-silencing system in soybean [J]. Sci Agric Sin, 2015, 48(12): 2479-2486.
28	Yan HX, Fu DQ, Zhu BZ, et al. Sprout vacuum-infiltration: a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species [J]. Plant Cell Rep, 2012, 31(9): 1713-1722.
29	Li XY, Tao N, Xu B, et al. Establishment and application of a root wounding-immersion method for efficient virus-induced gene silencing in plants [J]. Front Plant Sci, 2024, 15: 1336726.
30	郝梦媛, 杭琦, 师恭曜. VIGS基因沉默技术在作物基因功能研究中的应用与展望 [J]. 中国农业科技导报, 2022, 24(1): 1-13.
	Hao MY, Hang Q, Shi GY. Application and prospect of virus-induced gene silencing in crop gene function research [J]. J Agric Sci Technol, 2022, 24(1): 1-13.

[1]	李艳伟, 杨妍妍, 孙亚玲, 霍雨猛, 王振宝, 刘冰江. 基于转录组分析植物激素对洋葱鳞茎膨大发育的调控机制[J]. 生物技术通报, 2025, 41(2): 187-201.
[2]	许雪飞, 杨盼盼, 张文亮, 边光亚, 徐雷锋, 刘会超, 明军. 百合斑驳病毒在卷丹顶端分生组织中的分布[J]. 生物技术通报, 2024, 40(8): 212-220.
[3]	李亦君, 杨小贝, 夏琳, 罗朝鹏, 徐馨, 杨军, 宁黔冀, 武明珠. 烟草NtPRR37基因克隆及功能分析[J]. 生物技术通报, 2024, 40(8): 221-231.
[4]	侯文婷, 孙琳, 张艳军, 董合忠. 基因编辑技术在棉花种质创新和遗传改良中的应用[J]. 生物技术通报, 2024, 40(7): 68-77.
[5]	花子晴, 周静远, 董合忠. 双子叶植物下胚轴和顶端弯钩发育及其对出苗的调控机制[J]. 生物技术通报, 2024, 40(4): 23-32.
[6]	华炫, 田博雯, 周欣彤, 江梓涵, 王诗琦, 黄倩慧, 张健, 陈艳红. 旱柳SmERF B3-45的克隆及耐盐功能研究[J]. 生物技术通报, 2024, 40(12): 124-135.
[7]	张怡, 张心如, 张金珂, 胡利宗, 上官欣欣, 郑晓红, 胡娟娟, 张聪聪, 穆桂清, 李成伟. 小麦镉胁迫响应基因TaMYB1的功能分析[J]. 生物技术通报, 2024, 40(1): 194-206.
[8]	娄慧, 朱金成, 杨洋, 张薇. 抗、感品种棉花根系分泌物对尖孢镰刀菌生长及基因表达的影响[J]. 生物技术通报, 2023, 39(9): 156-167.
[9]	刘保财, 陈菁瑛, 张武君, 黄颖桢, 赵云青, 刘剑超, 危智诚. 多花黄精种子微根茎基因表达特征分析[J]. 生物技术通报, 2023, 39(8): 220-233.
[10]	张勇, 徐田军, 吕天放, 邢锦丰, 刘宏伟, 蔡万涛, 刘月娥, 赵久然, 王荣焕. 种植密度对夏播玉米茎秆质量和根系表型性状的影响[J]. 生物技术通报, 2023, 39(8): 70-79.
[11]	刘珍银, 段郅臻, 彭婷, 王童欣, 王健. 基于三角梅的病毒诱导基因沉默体系的建立与优化[J]. 生物技术通报, 2023, 39(7): 123-130.
[12]	李文辰, 刘鑫, 康越, 李伟, 齐泽铮, 于璐, 王芳. TRV病毒诱导大豆基因沉默体系优化及应用[J]. 生物技术通报, 2023, 39(7): 143-150.
[13]	杨洋, 朱金成, 娄慧, 韩泽刚, 张薇. 海岛棉与枯萎病菌的互作转录组分析[J]. 生物技术通报, 2023, 39(6): 259-273.
[14]	刘思佳, 王浩楠, 付宇辰, 闫文欣, 胡增辉, 冷平生. ‘西伯利亚’百合LiCMK基因克隆及功能分析[J]. 生物技术通报, 2023, 39(3): 196-205.
[15]	徐俊, 叶雨晴, 牛雅静, 黄河, 张蒙蒙. 菊花根状茎发育的转录组分析[J]. 生物技术通报, 2023, 39(10): 231-245.