韩帅, 靳芊芊, 殷楠, 张国君, 徐兴兴. 2024: 柽柳再生和转化体系的建立及瞬时转化TcSYP121基因功能分析. 南方农业学报, 55(3): 823-833. DOI: 10.3969/j.issn.2095-1191.2024.03.022
引用本文: 韩帅, 靳芊芊, 殷楠, 张国君, 徐兴兴. 2024: 柽柳再生和转化体系的建立及瞬时转化TcSYP121基因功能分析. 南方农业学报, 55(3): 823-833. DOI: 10.3969/j.issn.2095-1191.2024.03.022
HAN Shuai, JIN Qian-qian, YIN Nan, ZHANG Guo-jun, XU Xing-xing. 2024: Establishment of a system for regeneration and transformation of Tamarix chinensis Lour. and functional analysis on transient transformation of TcSYP121 gene. Journal of Southern Agriculture, 55(3): 823-833. DOI: 10.3969/j.issn.2095-1191.2024.03.022
Citation: HAN Shuai, JIN Qian-qian, YIN Nan, ZHANG Guo-jun, XU Xing-xing. 2024: Establishment of a system for regeneration and transformation of Tamarix chinensis Lour. and functional analysis on transient transformation of TcSYP121 gene. Journal of Southern Agriculture, 55(3): 823-833. DOI: 10.3969/j.issn.2095-1191.2024.03.022

柽柳再生和转化体系的建立及瞬时转化TcSYP121基因功能分析

Establishment of a system for regeneration and transformation of Tamarix chinensis Lour. and functional analysis on transient transformation of TcSYP121 gene

  • 摘要: 【目的】建立柽柳再生及遗传转化体系并分析瞬时转化柳树突触融合蛋白(TcSYP121)基因功能,为筛选柽柳分子抗性育种候选基因及盐碱土的改良和生态修复提供理论参考。【方法】通过单因素筛选最佳外植体、消毒处理、各阶段(初代、分化、增殖和生根)的培养基类型及激素浓度建立柽柳再生体系;通过正交试验分析影响转化体系的4个因素(预培养时间、侵染时间、共培养时间和农杆菌浓度);通过PCR扩增及电泳结果验证柽柳转化体系;瞬时转化TcSYP121基因获得过表达植株(OE)和病毒诱导的基因沉默植株(VI),进行表型观测、植物超氧阴离子染色液(NBT)和二氨基联苯胺法(DAB)染色并测定盐腺直径和盐腺密度、叶绿素含量、超氧化物酶(SOD)活性、过氧化物酶(POD)活性及TcSYP121基因相对表达量,分析TcSYP121基因的功能。【结果】最优外植体为柽柳嫩茎段,最适宜次氯酸钠(NaClO)消毒浓度为5%,最佳消毒时间为5 min,最适合的初代培养基为MS培养基,最佳分化培养基为MS+1.0mg/L 6-BA+0.1 mg/L NAA,最佳增殖培养基为MS+1.5 mg/L 6-BA+0.1 mg/L IBA,最佳生根培养基是1/2 MS+0.5mg/L IBA。转化体系中正交试验结果为最佳预培养时间是2 d,最佳侵染时间是5 min,最佳共培养时间是2 d,最佳农杆菌浓度OD600为0.8,抗生素头孢霉素(Cef)浓度为300 mg/L,卡那霉素(Kan)筛选压为30 mg/L。PCR扩增及电泳结果显示,利用该遗传转化体系可获得阳性转基因植株。瞬时转化TcSYP121基因柽柳的耐盐功能分析结果显示,在盐胁迫下,VI和对照(CK)柽柳中叶绿素含量显著下降(P<0.05),而OE下降不显著(P>0.05);OE柽柳SOD和POD活性均高于VI与CK,且OE中TcSYP121基因的相对表达量高于CK和VI。【结论】建立了柽柳的再生体系和遗传转化体系,通过对柽柳进行瞬时转化获得过表达和沉默表达柽柳,证实TcSYP121基因能够提高柽柳耐盐性。

     

    Abstract: 【Objective】To establish a regeneration system and genetic transformation system of Tamarix chinensis Lour. and analyze the function of transiently transformed T. chinensis syntaxin(TcSYP121) gene, in order to provide theoretical re-ferences for the screening of candidate genes of T. chinensis molecular resistance breeding and the improvement and ecological restoration of saline and alkaline soil. 【Method】T. chinensis regeneration system was established by univariate analysis of the selection of explants, disinfection treatment, medium type and hormone combination concentration at each stage (primary, differentiation, proliferation and rooting); four factors affecting the transformation system (preculture time, infestation time, co-culture time and Agrobacterium concentration) were analyzed by orthogonal test; and the transformation system of T. chinensis was established by the results of PCR amplification electrophoresis. Meanwhile, the transient transformation of TcSYP121 gene obtained overexpression plants (OE) and virus-induced gene silencing plants (VI), and phenotypic observation, plant superoxide anion dyeing solution(NBT) and diaminobenzidine method (DAB) staining, salt gland diameter and density, chlorophyll content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, and the relative expression of the TcSYP121 gene were determined to analyze the function of TcSYP121 gene. 【Result】The optimal explants were the tender stem segment of T. chinensis, the most suitable disinfection method was 5% sodium hypochlorite(NaClO) ,the best disinfection time was 5min, the most suitable primary medium was MS medium, and the best differentiation medium was MS+1.0 mg/L 6-BA+0.1 mg/L NAA. The best rooting medium was 1/2 MS+0.5 mg/L IBA. The results of orthogonal test in the transformation system were the best pre-culture time being 2 d, the optimal infection time being 5 min, the optimal co-culture time being 2 d, the optimal Agrobacterium concentration OD600 value being 0.8, the antibiotic concentration Cef being 300 mg/L, and kanamycin(Kan) screening pressure was 30 mg/L. PCR amplification electrophoresis results showed that positive transgenic plants could be obtained using this genetic transformation system. The results of salt tolerance function analysis of transiently transformed TcSYP121 gene of T. chinensis showed that under salt stress, the chlorophyll content in VI and control (CK) decreased significantly (P<0.05), and the OE decrease was not significant (P>0.05); OE T. chinensis had a higher level of SOD and POD activities than VI and CK, the relative expression of TcSYP121 gene in OE was higher than that in CK and VI. 【Conclusion】The regeneration system and genetic transformation system of T. chinensis were preliminarily established, and overexpressed and silenced T. chinensis were obtained through transient transformation of T. chinensis Lour., which confirmed that the TcSYP121 gene could improve the salt tolerance of T. chinensis.

     

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