Cloning，Expression and Functional Analysis of Potassium Channel Gene MiSPIK in Mangifera indica

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

Abstract

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

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.

Figures/Tables 11

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

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）

Fig. 2 Functional domain analysis

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

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

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

Fig. 6 Tissue specific expression analysis

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

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

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**

Fig. 9 Phenotype analysis of T1 transgenic lines

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