黑籽南瓜NBS类基因CfRFN2的克隆及VIGS验证
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国家自然科学基金(31460516);


Cloning and VIGS Functional Confirming of NBS Type Diseaseresistance Gene from Cucurbita ficifolia
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    摘要:

    黑籽南瓜(Cucurbita ficifolia)是云南特有的具有高抗枯萎病遗传性状的瓜类种质资源。为鉴定黑籽南瓜中NBSLRR类基因的抗病功能,该研究从其叶片中克隆了NBS类基因CfRFN2 (GenBank ID:MK618462),测序全长为4 303 bp,完整的编码框长度为4 092 bp,编码1 363个氨基酸残基,该基因注释为拟南芥抗病蛋白At4g27190类转录体X1的同源基因,含有1个NBARC和2个LRR结构域,属于具有信号肽的可溶性蛋白。核苷酸相似性分析显示,CfRFN2与其他瓜类NBS类基因相似性在87%~98%之间;系统进化树分析表明,CfRFN2 蛋白和瓜类的其他NBS类抗病蛋白聚为一个分支,其中CfRFN2 蛋白与中国南瓜和美洲南瓜的RPS2、印度南瓜的RPS2like亲缘关系最近,其次是黄瓜的At4g27190和苦瓜的At4g27220,与甜瓜的Atg27190亲缘关系相对较远;组织表达特性分析表明,CfRFN2基因在黑籽南瓜叶片中表达量最高,其次是茎,而在果皮和根中表达量较低。该研究采用烟草脆裂病毒载体系统,构建了黑籽南瓜VIGS沉默载体pTRV2CfRFN2,含沉默载体的农杆菌侵染黑籽南瓜幼苗后接种枯萎病菌,qRTPCR检测表明,接种后2 d 和4 d的转pTRV2CfRFN2沉默组植株的CfRFN2基因表达量比接种后同时期的野生型植株显著降低(分别下降34.75%和98.27%),病情指数增加为野生型的1.32倍,初步证明黑籽南瓜CfRFN2基因具有抗枯萎病的功能,推测该基因可能在黑籽南瓜抗枯萎病防御过程中发挥着重要作用。该研究中NBS类基因CfRFN2的克隆和VIGS验证为黑籽南瓜更多优异基因的克隆和功能验证奠定了前期基础,也为发掘黑籽南瓜优异抗病基因和开展瓜类分子育种提供新信息。

    Abstract:

    Cucurbita ficifolia Bouche. is a unique cucurbits germplasm resource in Yunnan Province with highly Fusarium wiltresistance genetic trait. To explore the function of NBSLRR type genes from C. ficifolia, in this study, we isolated the NBS type gene of CfRFN2 (GenBank ID:MK618462). We obtained a full length 4 303 bp by divided three fragment cloning using RTPCR method from leaves of C. ficifolia. The CfRFN2 gene was annotated as a homologous gene of transcriptionoid X1 of Arabidopsis diseaseresistant protein At4g27190, with complete coding frame length of 4 092 bp and encoding 1 363 amino acids, and its deduced protein contained one NBARC conserved domain and two LRR domains. The CfRFN2 gene belongs to a hydrophilic protein containing the signal peptide. The results of nucleotide similarity analysis indicated that the identity of CfRFN2 gene varied from 87% to 98% when compared with the other disease resistance genes in Cucurbitaceae crops. Phylogenetic analysis results showed that CfRFN2 protein and other NBS type protein from cucurbits crops were clusterd into one branch, indicating it had closer genetic relation among cucurbits crops than with other species NBS protein. Moreover, CfRFN2 had the closest genetic relation with RPS2 of Cucurbita pepo and Cucurbita moschata, Cucurbita maxima RPS2like protein, followed by Cucurbita sativus At4g27190 and Momordica charartia At4g27220, while relatively quite far from Cucurbita melo At4g27190. The tissue specific expression results indicated that the CfRFN2 gene was highly expressed in leaves than that in stems, and had low expression level in fruits and roots. Furthermore, based on the tobacco rattle virus vector system, we also constructed VIGS silencing vector pTRV2CfRFN2, constantly the seedlings of C. ficifolia were infected by Agrobacterium tumefaciens carrying silencing vector and inoculated with Fusarium oxysporum after 4 weeks infection (wild type plants inoculation pathogeny simultaneously). The qRTPCR analysis showed that, compared to the wild type plant, the expression of CfRFN2 gene in silencing plants decreased by 34.75% and 98.27%, respectively after 2 and 4 days inoculation. Moreover, the average disease index of silencing plants was 1.32 times that of wild type plants. This preliminarily results suggested that CfRFN2 gene in C. ficifolia had the function of resistance to Fusarium wilt. The cloning and VIGS confirmation of NBS type gene CfRFN2 would be the basis for digging more disease resistant genes in C. ficifolia, and thus provide a new clue for carrying out molecular breeding and exploring the disease resistant mechanism in cucurbits crops.

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丁玉梅,侯思名,姚春馨,等.黑籽南瓜NBS类基因CfRFN2的克隆及VIGS验证[J].西北植物学报,2021,41(5):727-737

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  • 在线发布日期: 2021-06-22
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