在中国大豆品种上创建ALSV诱导的基因沉默体系

doi:10.3864/j.issn.0578-1752.2022.09.002

中国农业科学 ›› 2022, Vol. 55 ›› Issue (9): 1710-1722.doi: 10.3864/j.issn.0578-1752.2022.09.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

在中国大豆品种上创建ALSV诱导的基因沉默体系

董永鑫¹(),卫其巍^1,²(),洪浩¹,黄莹¹,赵延晓¹,冯明峰¹,窦道龙^1,^*(),徐毅^1,^*(),陶小荣^1,^*()

¹南京农业大学植物保护学院/农业农村部大豆病虫害防控重点实验室,南京 210095
²四川省达州市农业科学研究院,四川达州 635000

收稿日期:2021-11-23 修回日期:2022-01-10 出版日期:2022-05-01 发布日期:2022-05-19
通讯作者: 窦道龙,徐毅,陶小荣
作者简介:董永鑫,E-mail： 2019102035@njau.edu.cn。|卫其巍,E-mail： 675815483@qq.com。
基金资助:
国家自然科学基金杰出青年基金(31925032);海南省重大科技计划(ZDKJ2021007)

Establishment of ALSV-Induced Gene Silencing in Chinese Soybean Cultivars

DONG YongXin¹(),WEI QiWei^1,²(),HONG Hao¹,HUANG Ying¹,ZHAO YanXiao¹,FENG MingFeng¹,DOU DaoLong^1,^*(),XU Yi^1,^*(),TAO XiaoRong^1,^*()

¹College of Plant Protection, Nanjing Agricultural University/Key Laboratory of Soybean Disease and Pest Control of Ministry of Agriculture and Rural Affairs, Nanjing 210095
²Dazhou Academy of Agricultural Sciences in Sichuan Province, Dazhou 635000, Sichuan

Received:2021-11-23 Revised:2022-01-10 Online:2022-05-01 Published:2022-05-19
Contact: DaoLong DOU,Yi XU,XiaoRong TAO

摘要/Abstract

摘要：

【目的】在中国大豆品种上建立以苹果潜隐球形病毒（apple latent spherical virus,ALSV）为载体的基因沉默体系,为中国大豆品种的基因功能和遗传育种提供一种简便、省时、易操作的技术体系。【方法】构建以农杆菌介导接种的ALSV病毒侵染性克隆载体。从大豆品种威廉姆斯82（Williams 82）中特异扩增327 bp的八氢番茄红素脱氢酶（GmPDS）基因cDNA片段,插入病毒载体pALSV2。通过农杆菌浸润法将病毒载体导入模式植物本氏烟（Nicotiana benthamiana）,17 d后富集病毒粒子,摩擦接种大豆第一轮真叶,以接种病毒空载作为对照组,持续观察测试大豆植株系统叶表型,并结合逆转录-聚合酶链式反应（RT-PCR）或荧光定量PCR（qRT-PCR）检测ALSV衣壳蛋白基因（CP）和GmPDS的表达水平。【结果】ALSV﹕GmPDS接种大豆品种威廉姆斯82和南农1138-2,20 d后,前者系统叶未见白化表型,而后者系统叶出现明显的白化表型;qRT-PCR检测结果表明,发生白化的南农1138-2植株中GmPDS的表达水平显著降低,未出现白化表型的威廉姆斯82中GmPDS的表达水平没有出现显著变化。在此基础上,采用相同的方法,测试了ALSV在其他9种大豆品种中诱导GmPDS的沉默效率,发现在南农47、安豆203、祥斗4号、中黄13、山宁29、齐黄34等大豆品种上接种ALSV﹕GmPDS后植株系统叶均产生白化表型,而菏豆12、中黄311和山宁16等3个品种的GmPDS均不能诱导有效沉默。【结论】构建了农杆菌介导的ALSV病毒载体,利用本氏烟扩繁富集ALSV病毒,将提纯的病毒粒体摩擦接种大豆真叶,在多个中国大豆品种上成功建立了基因沉默体系。

关键词: 病毒诱导的基因沉默, 大豆, 苹果潜隐球形病毒, 大豆八氢番茄红素脱氢酶基因

Abstract:

【Objective】 The establishment of apple latent spherical virus (ALSV)-induced gene silencing on domestic soybean cultivars will offer a simple, time-saving and operable system for gene function and genetic breeding of soybean cultivars in China.【Method】The infectious clones of ALSV which was used to infect plant through Agro-infiltration was constructed. A 327 bp cDNA fragment of phytoene desaturase (GmPDS) cDNA was amplified from soybean cultivar Williams 82 and inserted into the pALSV2 vector. The pALSV1 and pALSV2-GmPDS were used to co-infect Nicotiana benthamiana plant through Agro-infiltration, and the ALSV virus particle was purified from systemic infected leaves of N. benthamiana plants at 17 days post infiltration (dpi). The purified virions were mechanically inoculated onto the first-round true leaves of soybean and photobleaching phenotype on the upper non-inoculated leaves was monitored from 10 to 30 dpi. Using the empty ALSV vector as a control, the gene expression levels of ALSV coat protein (CP) gene and GmPDS in ALSV:GmPDS infected soybean were examined by RT-PCR and quantitative RT-PCR analysis, respectively. 【Result】At 20 dpi, soybean cultivar Nannong 1138-2 inoculated with ALSV:GmPDS showed photobleaching phenotype in the systemic infected leaves, however Williams 82 did not show gene silencing phenotype of PDS in the systemic infected leaves. The results of qRT-PCR confirmed that the RNA expression level of GmPDS in Nannong 1138-2 was significantly reduced but ALSV:GmPDS was unable to silence PDS in Williams 82. By using the same method, the gene silencing efficiency of other 9 domestic soybean cultivars mediated by ALSV:GmPDS was examined. Soybean cultivars including Nannong 47, Andou 203, Xiangdou 4, Zhonghuang 13, Shanning 29 and Qihuang 34 all displayed photobleaching phenotype in plant systemic infected leaves after inoculation with ALSV:GmPDS, while GmPDS was not silenced in Hedou 12, Zhonghuang 311 and Shanning 16.【Conclusion】In this study, an Agrobacterium-mediated ALSV viral VIGS vector was constructed. ALSV virions was propagated and purified from N. benthamiana plant. The purified virions were rub inoculated onto the first-round true leaves of soybean. Finally, an efficient gene silencing system was established on a number of domestic soybean cultivars in China.

Key words: virus-induced gene silencing (VIGS), soybean, apple latent spherical virus, phytoene desaturase (PDS)

董永鑫,卫其巍,洪浩,黄莹,赵延晓,冯明峰,窦道龙,徐毅,陶小荣. 在中国大豆品种上创建ALSV诱导的基因沉默体系[J]. 中国农业科学, 2022, 55(9): 1710-1722.

DONG YongXin,WEI QiWei,HONG Hao,HUANG Ying,ZHAO YanXiao,FENG MingFeng,DOU DaoLong,XU Yi,TAO XiaoRong. Establishment of ALSV-Induced Gene Silencing in Chinese Soybean Cultivars[J]. Scientia Agricultura Sinica, 2022, 55(9): 1710-1722.

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引物名称 <BOLD>P</BOLD>rimer name	序列 Sequence of the primers (5′-3′)	用途 Purpose
GmPDSF	CTGATTTCACACTCGAGCCCGCTGACATGTCAGTAACTTGC	GmPDS扩增 Amplification of GmPDS
GmPDSR	CCTTCTAGCAGGGATCCCCCCATTGAAGCTAAATATTTTTG	GmPDS扩增 Amplification of GmPDS
GmActinF	GATACTAGACTATCGTTCTCCTAACTCG	大豆内参引物（RT-PCR） Internal control primers (RT-PCR)
GmActinR	GCATGAAAATCAAGGTGGTTGC	大豆内参引物（RT-PCR） Internal control primers (RT-PCR)
qGmActinF	TAGAGCCACCAATCCAGACAC	大豆内参引物（qRT-PCR） Internal control primers (qRT-PCR)
qGmActinR	TCGTATGAGCAAGGAAATTGG	大豆内参引物（qRT-PCR） Internal control primers (qRT-PCR)
qGmPDSF	CCTGAACGGGTAACTGATGAGGTG	GmPDS沉默验证 Verification of the silence efficiency of GmPDS
qGmPDSR	CGGCATACAAAGTCTTTCGGGTG
ALSV2F	CTTGTGTATGATGAACTCATGCAATTG	ALSV CP引物 ALSV CP primers
ALSV2R	GAAGCTTAGGAAAGCGAGTGCG	ALSV CP引物 ALSV CP primers
1F	GCATAGATCTGACCCAAATCTGC	验证目的片段 Verification of the target fragment
1R	CTTTGTGAAATCAGGGCCCTGAC	验证目的片段 Verification of the target fragment
NbEF1αF	TGCTGCAACAAGATGGATGC	本氏烟内参引物 Internal control primers for Nicotiana benthamiana
NbEF1αR	CCAGAGATGGGGACAAAGGG

品种 Cultivar	ALSV系统侵染 Systemic infection of ALSV	系统叶变白 <BOLD>P</BOLD>hotobleaching in systemic leaves	白化植物数/接种植物数 No. of plants showing photo-bleaching/No. of inoculated plants
南农1138-2 Nannong 1138-2	√	√	6/6
威廉姆斯82 Williams 82	√	×	0/6
齐黄34 Qihuang 34	√	√	3/8
安豆203 Andou 203	√	√	3/8
南农47 Nannong 47	√	√	3/8
祥斗4号 Xiangdou 4	√	√	4/8
中黄13 Zhonghuang 13	√	√	4/6
山宁29 Shanning 29	√	√	4/6
山宁16 Shanning 16	√	×	0/6
菏豆12 Hedou 12	√	×	0/6
中黄311 Zhonghuang 311	√	×	0/6

在中国大豆品种上创建ALSV诱导的基因沉默体系

Establishment of ALSV-Induced Gene Silencing in Chinese Soybean Cultivars

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