Molecular Cloning Of Tonoplat Na~+/H~+ Transporter Gene BvNHX From Sugar Beet (Beta Vulgaris L.) And Construction Of Its Expression Vector

Soil salinization is one of the abiotic stresses that affect crop quality and yield.The tonoplat Na~+/H~+ antiporter plays an important role under salt stress in the survival of plants,can compartmentalize Na+ into the tonoplat to reduce the ion toxicity of cells,maintain the balance of ions and osmotic potential cells to improve plant salt tolerance.In this study,the full-length cDNA of the tonoplat Na~+/H~+ antiporter gene BvNHX was cloned from sugar beet(Beta vulgaris L.),On this basis,we constructed the overexpression vector and RNAi expression vector of the tonoplat Na~+/H~+ antiporter gene BvNHX.The specific results were as follows:(1)Compared with tetraploid(TY03410),diploid(TY03209)was accumulated more Na+ in the shoots and roots of 200-300 mmol/L NaCl,but the content of K+ was not significantly different,however,there was no significant difference in K+/Na+ between 50-300 mmol/L NaCl treatment,compared with tetraploid(TY03410),the relative growth rate of diploid(TD85ms)was higher under 100 mmol/L NaCl treatment,but no significant difference of K+/Na+ under 50-300 mmol/L NaCl,so there was no significant difference in salt tolerance between tetraploid and diploid;The dry weight,fresh weight and Na+ content of tetraploid(TY03410)and diploid(TD85ms)increased with prolonged treatment under 100 mmol/L NaCl,But there was no significant difference in K+/Na+ when the root was treated for 72 h and the shoot was treated for 120 h.(2)Based on the known plant tonoplat Na~+/H~+ antiporter NHX gene to designe the degenerate primers,a tonoplat Na~+/H~+ cDNA(BvNHX)gene was cloned from sugar beet by RACE,The cDNA is 2284 bp in length including an open reading frame(ORF)of 1653 bp,a 5' untranslated region(5'URT)of 19 bp and a 3' untranslated region(3'URT)of 612 bp with a ploy(A)tail.The BvNHX cDNA encodes a protein of 552 amino acids with a deduced molecular mass of 61.34 KDa and a pI of 6.609.BvNHX has twelve putative transmembrane(TM)domains,the Amiloride binding site LFFYLLPPI located in the third transmembrane region and has high homology with other species.(3)Through the analysis of the full length sequence of the tonoplat Na~+/H~+ antiporter gene,1653 bp was selected as the target sequence.The specific primers with cleavage site were designed and cloned by RT-PCR to construct the BvNHX overexpression vector pCAMBIA1302-BvNHX of BvNHX and transformed into Agrobacterium tumefaciens GV3101 by freeze-thawing method.(4)A highly conserved 500 bp fragment was selected as RNAi fragment by comparing the nucleotide sequence of tonoplat Na~+/H~+ antiporter gene of 23 species.The specific primers containing the cleavage site were designed to amplifiction the positive-sense strand and anti-sense strand to construct RNAi expression vector of the toloplast Na~+/H~+ antiporter gene BvNHX.Based on the intermediate vector pHANNIBAL and the expression vector pART27,the interference vector PARB of the BvNHX gene was successfully constructed by restriction enzyme digestion and ligation.(5)Using the method of seedling infestation to obtain the transgenic plants,PCR and RT-PCR was used to determine whether pARB was integrated into the plant genome and the BvNHX gene was silenced.The results showed that we obtained 12 BvNHX gene silence plants with different degrees.