柠条锦鸡儿CkPHB基因调控木质部发育增强植物抗旱性的功能分析
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国家自然科学基金(31770648,31070538);


Functional Analysis of CkPHB Gene Regulating Xylem Development and Enhancing Plant Drought Resistance in Caragana korshinskii
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    摘要:

    为了深入揭示柠条锦鸡儿(Caragana korshinskii)亮氨酸拉链蛋白转录因子(HDZIP Ⅲ)家族成员CkPHB的功能及其逆境响应,该研究参照转录组序列设计引物克隆得到CkPHB基因序列,并对其进行了一系列生物信息学分析,通过免疫组织化学染色方法对CkPHB蛋白在柠条锦鸡儿叶片中的组织定位进行观察,同时将克隆得到的CkPHB基因经农杆菌介导转化并筛选出阳性过表达拟南芥植株进行功能验证。结果表明:(1)成功克隆得到了柠条锦鸡儿中HDZIP Ⅲ家族成员CkPHB基因,其全长序列1 170 bp;CkPHB蛋白由389个氨基酸组成,分子量为42.83 kD,其编码的蛋白为疏水性蛋白,二级结构主要由α螺旋组成。(2)免疫组织化学分析显示, CkPHB蛋白定位于柠条锦鸡儿叶脉维管束中木质部区域。(3)经遗传转化筛选鉴定,最终获得了转柠条锦鸡儿CkPHB基因拟南芥过表达株系(CkPHBOE)T3代植株3株;解剖结构比较发现,与野生型拟南芥(WT)相比,拟南芥CkPHBOE株系的叶脉更为发达、维管束体积增大、木质部导管数量增加;实时定量PCR分析结果显示,拟南芥CkPHBOE株系中与木质部发育相关的5个基因(XCP2、CesA7、CesA8、PAL4、F5H)较WT均显著上调表达。(4)抗旱性实验结果显示,拟南芥CkPHBOE株系在干旱条件下存活率显著提升,生理指标的测定进一步支持上述结果,表明CkPHB过表达显著增强了转基因拟南芥的抗旱性。研究认为,过表达柠条锦鸡儿CkPHB基因的拟南芥输导组织更加发达、抗性生理指标显著提高,从而使抗旱能力增强,证明CkPHB基因在促进叶脉发育提高植物抗旱性中发挥重要作用。该研究结果为进一步研究柠条锦鸡儿CkPHB基因的干旱应答机制奠定了基础。

    Abstract:

    In order to reveal the function and stress response of CkPHB, we obtained a member of the leucine zipper protein transcription factor (HDZIP Ⅲ) family in Caragana korshinskii, CkPHB gene sequence by designing primer cloning according to transcriptome sequence, and carried out a series of bioinformatics analysis. The tissue localization of CkPHB protein in C. korshinskii leaves was observed by immunohistochemical staining. At the same time, the cloned CkPHB gene was transformed into Arabidopsis thaliana by Agrobacterium tumefaciensmediated method, and the positive overexpression plants were screened for functional verification. The results showed that: (1) the CkPHB gene, a member of the HDZIP Ⅲ family in C. korshinskii was successfully cloned. Its fulllength sequence is composed of 389 amino acids with a molecular weight of 42.83 kD. The protein encoded by CkPHB is hydrophobic and its secondary structure is mainly composed of αhelix. (2) Immunohistochemical analysis showed that CkPHB protein was localized in the xylem region of the vascular bundle of C. korshinskii leaves. (3) Through genetic transformation screening and identification, we finally obtained three T3 generation plants of C. korshinskii CkPHB gene overexpression line in A. thaliana (CkPHBOE). Compared with wildtype A. thaliana (WT), the CkPHBOE strain of A. thaliana has more developed veins, larger vascular bundle volume and more xylem vessels. The realtime quantitative PCR analysis showed that five genes related to xylem development in CkPHBOE strain (XCP2, CesA7, CesA8, PAL4, F5H) were upregulated compared with WT. (4) The results of drought resistance experiments showed that the survival rate of CkPHBOE strain in A. thaliana was significantly improved under drought conditions. The physiological indexes further supported the above results, indicating that CkPHB overexpression significantly enhanced the drought resistance of transgenic A. thaliana. The research shows that A. thaliana overexpressing C. korshinskii CkPHB gene is more developed and its physiological indexes of resistance are significantly improved, thus enhancing drought resistance, which proves that CkPHB gene plays an important role in promoting leaf vein development and improving plant drought resistance. The results of this study laid a foundation for further studying the drought response mechanism of CkPHB gene in C. korshinskii.

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李佳阳,谢丽芳,任洁洁,等.柠条锦鸡儿CkPHB基因调控木质部发育增强植物抗旱性的功能分析[J].西北植物学报,2021,41(8):1259-1266

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  • 在线发布日期: 2021-09-10
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