Identification Of Sugar Beet Shaker-type K~+ Channel And BvSKOR Function Analysis

Sugar beet（Beta vulgaris L.）is a biennial herb in the family Chenopodiaceae which is one of the very important crops in China’s food industry and sugar industry.The efficient absorption and utilization of K⁺by sugar beet helps promote its growth and stress tolerance.The Shaker family K⁺channel plays a key role in plants uptake and transport of K⁺,but there are few reports of the sugar beet Shaker channel.Given this,firstly,all members of the Shaker K⁺channel family were identified in sugar beet by bioinformatics analysis.Secondly,the expression pattern of BvSKOR,a gene encoding the outward rectifier K⁺channel,was analyzed by q RT-PCR method.Finally,overexpression vector of BvSKOR was constructed,and genetically transformed in tobacco to further analyze its function.The objective of this study was to enrich the potassium nutrient transport and stress resistance mechanism of sugar beet.The main results are as follows:（1）A total of 6 genes of Shaker-type K⁺channels,including 5 inward rectifier channels Bv KAT1,Bv KAT3,Bv AKT1,Bv AKT2,Bv AKT5 and only one outward rectifier channel BvSKOR were identified in the genome of sugar beet.Their CDS ranged in length from 2232 bp（Bv KAT3）to 2739 bp（Bv AKT5）and encoded 743-912 amino acids.As a transmembrane protein,Shaker-type K⁺channels contain 6 transmembrane structures,and the transmembrane region is relatively conserved.In particular,the PD domain of all Shaker-type K⁺channels contain an identical Tx GYGD conserved sequence.In addition,Shaker-type K⁺channel proteins are mainly regulated by the CIPK family,such as CIPK9,CIPK6,CIPK23,etc.Phylogenetic analysis showed that the Shaker-type K⁺channel was divided into two groups,in which the evolutionary relationship of the outward K⁺rectifier of all species was closer,but the relationship was farther between the inward and outward rectified K⁺channels.（2）BvSKOR is expressed in both the roots and shoots of sugar beet.In addition,the expression of BvSKOR was upregulated by PEG treatment,and the longer the stress time,the higher the expression.Na Cl can significantly increase the relative expression level of BvSKOR in a short time（3 h）,and the expression of genes increases with increasing concentration.The relative expression level of BvSKOR showed an inverted U-function with the stress time,in which the relative expression level gradually increased in the first 12 h,decreased slightly after24 h,but was still significantly higher than the control.（3）Total RNA was extracted from sugar beet,and c DNA was reverse transcribed as a template to clone the full CDS length of the BvSKOR gene.Transfer the cloned sequence to the vector p Earley Gate 100 into competent Escherichia coli.The p EG100-BvSKOR overexpression vector was successfully constructed after identification by colony PCR and sequencing identification.（4）Overexpression of BvSKOR transgenic tobacco was obtained by Agrobacterium-mediated method.T₁ generation seeds of genetically modified tobacco were identified by Basta resistance and PCR identification.Under salt stress and osmotic stress,the root system of transgenic tobacco was significantly longer than that of wild-type,and the relative water content,chlorophyll,osmotic modulator content and antioxidant enzyme activity were significantly higher than those of wild-type.These results indicated that the overexpression of BvSKOR improved the growth status and enhanced salt-and drought-tolerance in transgenic tobacco under stress conditions.In summary,BvSKOR plays an important role in the responses of sugar beet to salt-and drought-stress.Overexpression of BvSKOR significantly enhanced these stresses tolerance of transgenic tobacco.This study should provide some theoretical support and genetic resources for the genetic improvement of crop stress tolerance.