Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (9): 1710-1722.doi: 10.3864/j.issn.0578-1752.2022.09.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Establishment of ALSV-Induced Gene Silencing in Chinese Soybean Cultivars

DONG YongXin1(),WEI QiWei1,2(),HONG Hao1,HUANG Ying1,ZHAO YanXiao1,FENG MingFeng1,DOU DaoLong1,*(),XU Yi1,*(),TAO XiaoRong1,*()   

  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 E-mail:2019102035@njau.edu.cn;675815483@qq.com;taoxiaorong@njau.edu.cn;xuyiqdpd@njau.edu.cn;ddou@njau.edu.cn

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)

Table 1

Primers used in this study"

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
GmActinF GATACTAGACTATCGTTCTCCTAACTCG 大豆内参引物(RT-PCR)
Internal control primers (RT-PCR)
GmActinR GCATGAAAATCAAGGTGGTTGC
qGmActinF TAGAGCCACCAATCCAGACAC 大豆内参引物(qRT-PCR)
Internal control primers (qRT-PCR)
qGmActinR TCGTATGAGCAAGGAAATTGG
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
1F GCATAGATCTGACCCAAATCTGC 验证目的片段
Verification of the target fragment
1R CTTTGTGAAATCAGGGCCCTGAC
NbEF1αF TGCTGCAACAAGATGGATGC 本氏烟内参引物
Internal control primers for Nicotiana benthamiana
NbEF1αR CCAGAGATGGGGACAAAGGG

Fig. 1

Construction of pALSV2-GmPDS silencing vector A: Schematic diagram of pALSV2-GmPDS construction. P35S: Enhanced CaMV 35S promoter; Tnos: Nopaline synthase terminator; PRO-co: Protease cofactor; HEL: NTP-binding helicase; C-PRO: Cysteine protease; POL: RNA polymerase; MP: 42K movement protein; Vp25, Vp20, and Vp24: Capsid proteins. B: Sequence alignment of GmPDS cDNA amplified regions"

Fig. 2

The procedure of ALSV virus particle extraction and enrichment from infected Nicotiana benthamiana plants"

Fig. 3

Phenotypes and RT-PCR detection of ALSV in systemic leaves of Nicotiana benthamiana by Agroinfitration A: Agrobacterium-mediated ALSV infection of N. benthamiana plant. Phenotypes of N. benthamiana at 17 d after infiltrated with 1﹕1﹕1 mixture of agrobacteria containing pALSV1, pALSV2-derivative vectors or P19. MOCK: Infiltrated with empty vector; P19: Gene silencing suppressor; B: The content of ALSV CP in systemic leaves was detected by RT-PCR; C: Agrobacterium-mediated ALSV﹕GmPDS infection of N. benthamiana plant and effect of P19 on ALSV infection; CH42: Chlorophyll synthesis related genes; D: The ALSV CP and inserted CH42 or GmPDS sequences in systemic leaves of plants in panel C was detected by RT-PCR"

Table 2

Gene silencing efficiency in eleven soybean cultivars using ALSV induced VIGS"

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

Fig. 4

Phenotypes and qRT-PCR detections of Nannong 1138-2 and Williams 82 mechanically inoculated with ALSV:GmPDS virus particle A: Phenotypes of Nannong 1138-2 mechanically inoculated with ALSV virus particles at 20 days post inoculation (dpi); B: Phenotypes of Nannong 1138-2 inoculated with ALSV:GmPDS virus particles at 20 dpi; C: The phenotypes of systemic leaves of Nannong 1138-2 soybean cultivar treated with ALSV and ALSV:GmPDS virus particles from Figure A and B; left leaves were inoculated with ALSV virus particles, and right leaves were inoculated with ALSV:GmPDS virus particles; D: qRT-PCR was used to quantify GmPDS silencing efficiency in systemic leaves of Nannong 1138-2 soybean cultivar treated with ALSV and ALSV:GmPDS virus particles (n=3, **: P<0.01); E: Phenotypes of Williams 82 inoculated with ALSV:GmPDS virus particles at 20 dpi; F: qRT-PCR was used to detect relative RNA expression of GmPDS in systemic leaves of Williams 82 treated with ALSV and ALSV:GmPDS virus particles (n=3, ns: P>0.05)"

Fig. 5

Detection of ALSV accumulation in systemic leaves of Williams 82 +: Using cDNA from ALSV systemically infected leaves of Nannong 1138-2 showing photobleaching phenotype as a positive control; —: cDNA from healthy soybean as a negative control"

Fig. 6

Determination of virus induced gene silencing efficiency in nine different domestic soybean cultivars using ALSV:GmPDS Phenotypes of nine different soybean cultivars mechanically inoculated with ALSV:GmPDS virus particles at 20 dpi; ALSV:GmPDS treated Qihuang 34, Nannong 47, Xiangdou 4, Zhonghuang 13, Shanning 29 and Andou 203 all showed leave photobleaching phenotype; Shanning 16, Hedou 12 and Zhonghuang 311 did not show photobleaching phenotype"

Fig. 7

Detection of ALSV accumulation in systemic leaves of different soybean cultivars A: The accumulation of CP could be detected in the systemic leaves of Qihuang 34 inoculated with ALSV virus particles, and CP gene could be detected in three out of eight samples inoculated with ALSV:GmPDS; B: CP could not be detect or detected only with a very low accumulation in Hedou 12 inoculated with ALSV and ALSV:GmPDS, respectively; C: CP could only be detected with a very low accumulation in Zhonghuang 311 inoculated with ALSV and ALSV:GmPDS; D: CP could only be detected with a very low accumulation in Shanning 16 inoculated with ALSV and ALSV:GmPDS, respectively. +: Using cDNA from ALSV systemically infected leaves of Nannong 1138-2 showing photobleaching phenotype as a positive control. —: cDNA from healthy soybean as a negative control"

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