杧果钾通道基因MiSPIK的克隆、表达与功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0055

摘要/Abstract

摘要：

Shaker类型钾通道在植物生长发育中起重要作用，其在果树中的生物学功能依然未知。克隆并揭示杧果SPIK钾通道基因的功能，为研究热带果树Shaker类型钾通道的生物学功能提供基因资源和理论基础。以杧果品种‘桂热82’为材料，利用RACE-PCR技术克隆Shaker类型钾离子通道基因MiSPIK（GenBank ID：OM179914），利用生物信息学手段分析该基因及编码蛋白的序列特征，通过实时荧光定量PCR（RT-qPCR）分析该基因的组织特异性表达特征，及其在转录水平对缺钾、高钾、低温、NaCl和PEG处理等5种非生物胁迫的响应情况，并创制MiSPIK超表达转基因拟南芥株系。结果显示，MiSPIK蛋白的分子量为90 006.83 kD，等电点（pI）为7.59，含有6个跨膜结构，属于不稳定的亲水蛋白。杧果MiSPIK与梨PbrSPIK和拟南芥AtSPIK的氨基酸序列一致性高达59.46%，三者紧密聚为一类，遗传距离最近。在转录水平上，MiSPIK在杧果花粉中特异性表达，并在一年生嫁接苗根部受缺钾或NaCl处理的抑制均显著降低，受高钾、PEG或低温胁迫诱导而显著增强。MiSPIK在拟南芥中表达后增强了转基因株系的钾素富集能力，并促进转基因植株提早抽薹和开花。杧果MiSPIK是花粉中特异表达的Shaker类型钾通道基因，其表达水平易受多种非生物胁迫的调控，可能在杧果钾素营养高效与利用中发挥作用。

关键词: 杧果, Shaker类型钾离子通道, SPIK, 花粉, 异源超表达

Abstract:

Shaker-type potassium（K⁺）channels play important roles in plant growth and development. However，their biological function in fruit crops is still unknown. We focused on the cloning and functional verification of SPIK channel gene in mango（Mangifera indica），which provide gene resources and theoretical foundation for the biological function analysis of Shaker type K⁺ channels in tropical fruit plants. The Shaker type K⁺ channel gene MiSPIK（GenBank ID：OM179914）was isolated by RACE-PCR technology from ‘Guire 82’ mango. The sequence characteristics of this gene and its coding protein were analyzed via bioinformatics. The tissue-specific expression characteristics and its responses to 5 abiotic stresses，including K⁺deficiency，high K⁺ stress，low temperature，NaCl and PEG treatment，were further detected in the transcriptional level via qRT-PCR. MiSPIK-overexpressed transgenic Arabidopsis line was created. Results showed that MiSPIK protein had a molecular weight of 90 006.83 kD with the isoelectric point（pI）of 7.59，which possessed 6 transmembrane domains and belonged to unstable hydrophilic protein. The amino acid sequence identity was 59.46% among MiSPIK，pear PbrSPIK，and Arabidopsis AtSPIK，and phylogenetic tree analysis demonstrated that these three SPIK homologs were tightly clustered together and had the closest genetic distance. In the transcriptional level，MiSPIK was specifically expressed in mango pollens，and its expression in the roots of annual grafted seedlings significantly reduced by K⁺deficiency or NaCl treatment，and significantly enhanced by high K⁺stress，PEG or low temperature treatment. In addition，over-expression of MiSPIK in Arabidopsis significantly enhanced the K⁺ accumulation status and induced earlier bolting and flowering of transgenic plants. MiSPIK was a pollen-specifically expressed Shaker type K⁺channel gene in mango，whose expression was prone to be regulated by distinct abiotic stresses，and may take part in the high efficient utilization of K⁺ nutrition in mango.

Key words: Mangifera indica, Shaker type K⁺ channel, SPIK, pollen, heterogenous expression

韩蕾, 李俊林, 高爱平, 黄建峰, 李建召, 宋志忠. 杧果钾通道基因MiSPIK的克隆、表达与功能分析[J]. 生物技术通报, 2022, 38(10): 164-172.

HAN Lei, LI Jun-lin, GAO Ai-ping, HUANG Jian-feng, LI Jian-zhao, SONG Zhi-zhong. Cloning，Expression and Functional Analysis of Potassium Channel Gene MiSPIK in Mangifera indica[J]. Biotechnology Bulletin, 2022, 38(10): 164-172.

图/表 11

表1 本文所用引物序列

Table 1 Primers used in study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
GP1-F	TCTTCACCCCTCTGGA（A/G）TT（C/T）GG
GP1-R	GAGGGGACCGGCC（A/G）TC（A/G）TA（A/C）TC
GP2-out	CTAATGGTTAATACCAGCAATC
GP2-in	CAGAAGGAAGTGTTCCTCTATG
GP3-out	GCTGGTGCAGCCCTTCTGAATCAG
GP3-in	GACGTAACATACTTGACCCAC
MiSPIK-F	CCAGTCTAGGCAACAAACCAAG
MiSPIK-R	CGCCTCCATTGGCTATCAGATCTG
pHB-MiSPIK-F	GCCGAGCTCATGAAAATAACGTGGCTGAGAA
pHB-MiSPIK-R	GCGTCTAGATTACAACCCACCATGAGTTTTAC
MiActin-F	GAGGAGAAACAGAAGCAAGT
MiActin-R	AATCATCTTGCTTCTCACCC

图1 MiSPIK RACE克隆过程 A：保守区片段扩增（M：DL2000 marker；1：保守区扩增产物）；B：末端RACE扩增（M：DL2000 marker；3：3'-RACE扩增产物；4：5'-RACE扩增产物）；C：全长CDS扩增（M：DL2000 marker；1：CDS扩增产物）

Fig. 1 RACE cloning process of MiSPIK A：Amplification of conserved domain（M：DL2000 marker. 1：PCR product of conserved domain）. B：RACE PCR amplification of both ends（M：DL2000 marker. 3：RACE PCR product of 3'-end. 4：RACE PCR product of 5'-end）. C：Amplification of full length CDS（M：DL2000 marker. 1：PCR product of CDS）

图2 功能域分析

Fig. 2 Functional domain analysis

图3 24种植物Shaker类型通道同源蛋白系统发育树建立

Fig. 3 Phylogenetic tree construction of Shaker type chan-nel homologs from 24 plant species

图4 3种植物SPIK同源蛋白氨基酸序列一致性分析

Fig. 4 Identity analysis of amino acid sequence of SPIK homologs from 3 plant species

图5 3种植物SPIK同源蛋白三级结构预测

Fig. 5 Tertiary structure prediction of SPIK homologs from 3 plant species

图6 组织特异性表达分析

Fig. 6 Tissue specific expression analysis

图7 MiSPIK对非生物胁迫差异响应的热图分析

Fig. 7 Heat map analysis of differential responses of MiS-PIK to different abiotic stresses

图8 异源超表达转基因创制 A：重组表达载体构建示意图；B：T0代转基因株系PCR鉴定

Fig. 8 Generation of heterologous over-expression transg-enic seedlings A：Schematic diagram of recombinant expression vector construction. B：PCR identification of T0 transgenic lines

表2 T1代转基因株系生理指标分析

Table 2 Physiological indicator analysis of T1 transgenic lines

指标Indicator	对照Control	T₁转基因株系T₁ transgenic lines
总鲜重Total fresh weight/g	11.73±1.34	16.86±1.38**
地上部鲜重Fresh weight of shoots/g	10.53±1.12	15.59±1.09**
根部鲜重Fresh weight of roots/g	1.20±0.09	1.27±0.13
总根长Total root length/cm	19.26±0.21	19.34±0.23
地上部钾含量K⁺ concentration of shoots/（g·kg^-1 DW）	33.47±2.89	42.56±3.32*
根部钾含量K⁺ concentration of roots/（g·kg^-1 DW）	22.18±1.65	29.26±2.14**

图9 T1代转基因株系表型分析

Fig. 9 Phenotype analysis of T1 transgenic lines

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