Gene Editing And Functional Analysis Of BpHKT1 In Betula Platyphylla

Asian white birch（Betula platyphylla Suk.Hara）is a deciduous tree mainly distributed in the north temperate zone,and it is also a pioneer tree species in Northeast China.The wood is a yellowish-white,smooth texture,and has excellent wood properties,which can be widely used as construction and industrial wood and has high economic value.The triterpenes such as betulinol and betulinic acid in birch bark can be used in the manufacture of anti-tumor and anti-HIV drugs,with corresponding medicinal value,and can also be used in the manufacture of cosmetics and spices.As an important pioneer tree species and constructive species in the northern forest,the ecological value of birch has become increasingly prominent with the proposal of the double-carbon strategy.Birch shows sensitive under salt stress.With the expansion of saline-alkali land in Northeast China,how to restore saline-alkali land through an effective biological way is also an urgent problem to be solved.It is a feasible method to use a forest system to restore saline-alkali land.As a pioneer tree species,birch has the natural advantage of ecological restoration.If it can improve the salt tolerance of birch,then it will be a more effective restoration tree species in saline-alkali soil and exert its economic and ecological effects.HKT gene has been proved to function on plant salt tolerance in many plants,but its specific function in birch response to salt is not clear.Therefore,in this study,the BpHKT1 mutant of birch was constructed by CRISPR/Cas9,the phenotype of the mutant was analyzed in to further analyze the function of BpHKT1.The main results of this study are as follows:1.Bioinformatics analysis shows that there is only one copy of BpHKT1 gene in Betula platyphylla with the full-length 1494 bp of CDS and encodes 498 amino acids and has Tr KH domain.BpHKT1 protein belongs to the first group of HKT proteins according to the classification principle and is mainly located on the cell membrane.BpHKT1 protein has eight typical transmembrane domains.Phylogeny analysis showed that BpHKT1 protein was closely related to poplar Pt HKT1;2 protein.BpHKT1 was mainly expressed in roots and was significantly induced under salt stress.2.Two sg RNA were designed on the first exon of the BpHKT1 gene of Betula platyphylla,and two gene editing vectors were constructed by CRISPR/Cas9 technology,targeting the BpHKT1 gene.The vector was constructed by p SC1 plasmid and successfully transferred into Agrobacterium tumefaciens.Betula platyphylla BpHKT1 gene was edited by Agrobacterium tumefaciens-mediated mature zygotic embryo genetic transformation method.A total of 50mutants were detected in T₀generation.Mutant types include biallelic mutations（biallele）,heterozygous mutations and chimera types.Among them,the nucleotide mutation of H1-82leads to the early termination of the coding amino acid,which can be used as a homozygous mutant.3.The plant height of bphkt1 mutant was significantly higher than that of wild type,and the photosynthetic efficiency of mutant was significantly lower than that of wild type under salt stress,and the contents of Na⁺,K⁺,and Na⁺/K⁺in different tissue parts of bphkt1 mutant shows that:Na⁺was significantly accumulated in the leaves of bphkt1 mutant under stress,the content of Na⁺in the root of bphkt1 mutant leaf was significantly lower than that of wild type under salt stress.BpHKT1 plays an important role in restricting the upward flow of Na⁺into leaves.Under salt stress,the expression patterns of transcription factors ERF,MYB,NAC and GPR,SOS1 genes related to salt stress in bphkt1 mutant were different from those in wild type,suggesting that BpHKT1 gene plays an important role in salt tolerance.