Construction and Validation of the TRV-mediated Gene Silencing System in Haloxylon ammodendron

doi:10.13560/j.cnki.biotech.bull.1985.2025-0527

Abstract

Abstract:

Objective To establish a virus-induced gene silencing (VIGS) system for Haloxylon ammodendron and verify its effectiveness. Method Using H. ammodendron seedlings, the CLA1 (Cloroplastos alterados 1) gene was selected as the indicator gene for constructing the recombinant virus vector, which was transformed into Agrobacterium GV3101. The root absorption method was employed to infect the roots of four-week-old seedlings, and the effects of different infection solutions on gene silencing induced by tobacco rattle virus (TRV) were evaluated. The system was also utilized to assess the role of the HaNAC3 gene in the positive regulation of salt stress. Result The albino phenotype in H. ammodendron seedlings was successfully induced using both the Agrobacterium-mediated root absorption method and the tobacco homogenate-mediated root absorption method. The silencing efficiency was 57.93% and 77.66% respectively. Compared to the control group, the relative expression of the HaCLA1 gene significantly reduced, with the tobacco homogenate infection showing a more pronounced silencing effect, thereby establishing the H. ammodendron VIGS system. Furthermore, the system was utilized to silence the HaNAC3 gene, serving as a positive control, resulting in a significant reduction in salt tolerance in gene-silenced H. ammodendron plants compared to the control group. Conclusion A preliminary VIGS system for H. ammodendron is successfully established, laying the groundwork for further studies on gene functionality related to stress responses.

Key words: Haloxylon ammodendron, virus-induced gene silencing, HaCLA1 gene, NAC transcription factor

SANG Shi-bo, LI Li, ZHANG Feng-yuan, SUN Lang, SHEN Neng-shuang, CHENG Cong, REN Yan-ping, MA Li, ZHANG Hua. Construction and Validation of the TRV-mediated Gene Silencing System in Haloxylon ammodendron[J]. Biotechnology Bulletin, 2025, 41(11): 212-220.

Figures/Tables 7

Table 1 Primers used in the study

引物名称 Name of primer	引物序列 Sequence of primer （5′-3′）	用途 Application
pTRV2-HaCLA1-F	GTGAGTAAGGTTACCGAATTCAGGAAGACCTCAGGGCTAGCA	沉默载体构建 Silent vector construction
pTRV2-HaCLA1-R	GAGACGCGTGAGCTCGGTACCCGTCCACTGGACCAATGTAGTACA
pTRV2-HaNAC3-F	TGAGTAAGGTTACCGAATTCGTACTACGAGGGAAAATATCCATCATC
pTRV2-HaNAC3-R	GAGACGCGTGAGCTCGGTACCTCATCTTGATCATCATTGTCTTCCTC
18SrRNA-F	CTCTGCCGTTGCTCTGATGAT	基因相对表达量分析 Analysis of relative gene expressions
18SrRNA-R	CCTTGGATGTGGTAGCCGTTC
RT-qPCR-HaNAC3-F	ATCAAAACAGCCAGATGTCA
RT-qPCR-HaNAC3-R	GAAGAAAGCTCCACTTCGTTG
RT-qPCR-HACLA1-F	CTTTAGGGAAGCTCCAAGCATT
RT-qPCR-HACLA1-R	CGCAACCTTGTGTGTTTGTCC

Fig. 1 Schematic diagram of pTRV1, pTRV2, pTRV2-HaCLA1and pTRV2-HaNAC3 vectorsLB: Left border. RB: Right border. 35S: CaMV 35S promoter. R: Self-cleaving ribozyme. NOSt: NOS terminator. CP: Viral capsid protein

Fig. 2 Flow chart of TRV virus-mediated VIGS in H. ammodendron

Fig. 3 Agarose gel electrophoresis verification of gene silencing fragment cloneM: 2 000 DNA marker. 1: HaCLA1 gene silencing fragment; 2: HaNAC3 gene silencing fragment

Fig. 4 Recombinant viral vector double enzyme digestion and verificationM:10 000 DNA marker;1:double digestion of pTRV2-HaCLA1 vector;2:pTRV2-HaCLA1 original plasmid electrophoresis

Fig. 5 Phenotype of HaCLA1 gene silencing and RT-qPCR analysis under different infection solutions (12 d of infestation)A:Whole-plant silencing phenotype of H. ammodendron after HaCLA1 gene silencing. B:Assimilating branch silencing phenotype of H. ammodendron after HaCLA1 gene silencing. C: Results of RT-qPCR analysis of HaCLA1 gene (12 d of infestation). ***P<0.001

Fig. 6 Application of the H. ammodendron VIGS systemA: Phenotypic changes of HaNAC3 gene silencing in H. ammodendron. (TRV:00:Empty vector negative control group;TRV2:HaCLA1: Positive control group;TRV2:HaNAC3: Experimental group). B: Relative expression of the HaNAC3 gene in H. ammodendron after 12 d and 30 d. C: Phenotypic characteristics of H. ammodendron plants under normal conditions and after 12 d of salt stress. D: Relative expressions of the HaNAC3 gene in H. ammodendron under normal conditions and after 12 d of salt stress. ** indicates significant differences at the P<0.01, and *** indicates significant differences at the P<0.001

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