过表达C2H2型锌指蛋白基因PdbZFP26提高山新杨耐盐性

doi:10.11707/j.1001-7488.LYKX20210971

摘要/Abstract

摘要：

目的: 土壤盐渍化是制约农林资源生产力的主要因素之一。为适应环境，植物会通过启动基因表达及生理和形态结构的变化来减缓盐害。越来越多的研究表明，C2H2型锌指蛋白在植物应答盐胁迫调控网络中扮演重要角色。迄今，关于植物C2H2锌指蛋白生物学功能的研究集中于植物特有的Q型，而对于非Q型C2H2锌指蛋白的认识非常有限。前人从毛果杨中鉴定到109个C2H2锌指蛋白的编码基因PtrZFPs，其中62个编码的蛋白为非Q型锌指蛋白。鉴定、解析耐盐相关C2H2锌指蛋白基因的功能将为深入理解植物应对非生物胁迫的分子机制提供重要资料。通过分析过表达PdbZFP26转基因山新杨的表型以鉴定其功能，本文以期为山新杨抗盐遗传改良提供优良资源及理论基础。方法: 本研究利用实时定量PCR方法分析PdbZFP26的组织器官表达模式及对非生物胁迫和植物激素的响应模式；利用叶盘转化法获得过表达PdbZFP26转基因山新杨，观察转基因和对照株系在盐胁迫处理前后的长势，测定其抗逆生理指标，比较不同株系对盐胁迫的耐受能力。结果: 基于前期表达谱数据分析，从山新杨茎中筛选到一个受盐胁迫诱导表达的C2H2锌指蛋白基因PdbZFP26，该基因编码区为2 061 bp，共编码686个氨基酸。PdbZFP26仅含有一个C2H2锌指结构域，且该结构域中不具备Q型特征基序“QALGGH”。进化树分析显示，PdbZFP26为C型C2H2型锌指蛋白。表达模式分析结果显示，PdbZFP26在茎中特异表达，主要集中在木质部(包含维管形成层)；除盐胁迫之外，ABA和BRs会引起PdbZFP26不同程度的上调，其中ABA胁迫诱导的上调幅度最为显著。转基因山新杨表型分析结果显示，盐胁迫条件下，过表达PdbZFP26转基因山新杨的生长明显优于对照，转基因株系的叶绿素含量显著高于对照，MDA和H₂O₂含量明显低于对照，而SOD、POD、CAT等抗氧化酶活性高于对照。结论: 过表达PdbZFP26可通过增加抗氧化物酶活性来降低过氧化物质的积累，从而提高转基因山新杨对盐胁迫的耐受性。

关键词: 山新杨, C2H2锌指蛋白, 盐胁迫

Abstract:

Objective: Soil salination is one of the main factors restricting the productivity of agricultural and forestry resources. To adapt to the environment, plants alleviate salt damage by initiating gene expression, altering the physiological metabolism and morphological structure. A growing number of studies have shown that C2H2 zinc finger proteins (ZFPs) play important roles in the regulatory networks of plant response to salt stress. To date, studies on the biological function of plant ZFPs have focused on plant-specific Q-type, while researches on non-Q-type ZFPs is very limited. Previously, 109 genes encoding C2H2 ZFPs, PtrZFPs, were identified from Populus trichocarpa, 62 genes of which encoded the proteins of non-Q-type ZFPs. Identification and analysis of the function of salt-resistant C2H2 zinc finger protein genes will provide important information for further understanding the molecular mechanisms of plants in response to abiotic stress. By analyzing the phenotypes and identifying the functions of transgenic P. davidiana×P. bolleana overexpressing PdbZFP26, this study aims to provide excellent resources and theoretical basis for genetic improvement of salt resistance of P. davidiana×P. bolleana. Method: In this study, real-time quantitative PCR was used to analyze the expression patterns of PdbZFP26 in different organs, and the response to abiotic stresses and phytohormones. The transgenic P. davidiana×P. bolleana was obtained by leaf disc transformation. The growth of transgenic and control lines was observed before and after salt stress treatment, and the physiological indexes of stress resistance were measured to compare the tolerance of different lines to salt stress. Result: Based on the analysis of preliminary expression profile data, a salt stress-inducible C2H2 zinc finger protein gene PdbZFP26 was screened from the stem cDNAs. The coding region of this gene is 2 061 bp, encoding 686 amino acids. PdbZFP26 contained only one C2H2 zinc finger domain and did not have the type-Q characteristic motif "QALGGH". The phylogenetic tree revealed that PdbZFP26 belonged to type-C type ZFPs. The expression pattern analysis showed that PdbZFP26 was specifically expressed in the stem, mainly in xylem (including vascular cambium); In addition to salt stress, both ABA and BRs were able to cause the upregulation of PdbZFP26 to different degrees. The result of the phenotypes showed that the transgenic P. davidiana×P. bolleana overexpressing PdbZFP26 grew significantly better than the control under salt stress, with significantly higher chlorophyll content, lower MDA and H₂O₂ content, and higher antioxidant enzyme activities such as SOD, POD and CAT. Conclusion: The overexpression of PdbZFP26 can reduce peroxide accumulation by increasing activities of antioxidases, and thus improve the tolerance of transgenic P. davidiana×P. bolleana to salt stress.

Key words: P. davidiana×P. bolleana, C2H2 zinc finger protein, salt stress

中图分类号:

S718.43

马姗姗,杨静静,曲德辉,李梦婕,张进,吴凡林,宿红艳,王磊. 过表达C2H2型锌指蛋白基因PdbZFP26提高山新杨耐盐性[J]. 林业科学, 2023, 59(1): 110-118.

Shanshan Ma,Jingjing Yang,Dehui Qu,Mengjie Li,Jin Zhang,Fanlin Wu,Hongyan Su,Lei Wang. Overexpression of PdbZFP26, a Gene Encoding C2H2 Zinc Finger Protein, Improves Salt Tolerance of Transgenic Populus davidiana×P. bolleana[J]. Scientia Silvae Sinicae, 2023, 59(1): 110-118.

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用途 Application	引物名称 Primer name	引物序列 Sequence (5′—3′)
载体构建 Vector construction	p2301-PdbZFP26-F	CGGGATCCATGCCAGTTGTGAAACCTGTGA
载体构建 Vector construction	p2301-PdbZFP26-R	GCGTCGACTTAAATGATCGTTCTTTGTTTCGG
基因组扩增 Genome amplification	P35S	TCATTTGGAGAGAACACGGGG
基因组扩增 Genome amplification	Pzfp26-3	CCAAAAGGTCAGAACCAGC
实时荧光定量PCR qRT-PCR	PActin-F	TCATCGGAATGGAAGCTGCTGGTA
	PActin-R	TTAAATGATCGTTCTTTGTTTCGG
	Pzfp26-1	GCCATTGCTGCTGAACATGTG
	Pzfp26-2	AGCTACATTCGCAAGTCCAC
	Pzfp26-4	GAGCCCGAGCTGATTACCTG
	Pzfp26-5	CTAGTCCTCACCCTTCCCAT

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