Cloning And Functional Research Of StRbohA And StRbohB Genes In Potato

Potato（Solanum tuberosum L.）,an annual herb of Solanaceae,is an important food and vegetable crop after wheat,rice and corn.The length of the tuber dormancy period is vital for the cultivation,production and further intensive processing of potato.Previous studies have shown that reactive oxygen species（ROS）play an important regulatory role in the dormancy and germination of potato tuber.Respiratory burst oxidase homologue（Rboh）,also known as NADPH oxidase,can cause the production of superoxide anion（O₂^-·）and H₂O₂ by catalyzing extracellular O₂.The St RbohA and St Rboh B gene coding sequences and their corresponding promoters were cloned and their gene functions were studied in this experiment.The main results are as follows:1.The complete CDS of St RbohA and St Rboh B genes were coloned with the length of2801bp and 2604bp,respectively.Overexpression vectors p BI121-St RbohA,p BI121-St Rboh B and RNAi interference vectors p Bin19-St RbohA and p Bin19-St Rboh B were constructed,respectively.The tubers of―Wh-15‖were infected withAgrobacterium infection method,and the transgenic plants were identified by qRT-PCR.Then,we obtained3 and 4 potato transgenic plants with overexpressed St RbohA and St Rboh B respectively.Besides,3 and 2 potato transgenic plants of RNAi interference were obtained respectively.Compared with wild type plants,over-expression St Rboh B gene transgenic seedlings showed different phenotype of lower plant and reducting leaf area.2.The expression patterns of St RbohA and St Rboh B genes in potato vegetative organs were analyzed by qRT-PCR.The expression levels of StrbohA gene in roots,stems,leaves and tubers were leaves,stems,roots and tubers from high to low,while the expression levels of Strboh B gene from stem to leaf,leaf,root and tubers were from high to low.Under normal growth conditions,St RbohA and St Rboh B genes were least expressed in tubers.After treated with 100μM ABA and GA₃ for 24 h,we found that the expression of StrbohA gene in roots increased and the expression of St Rboh B gene in roots and tubers increased after ABA treatment.After GA₃ treatment,the expression of St RbohA gene in roots and tubers increased,while the expression of St Rboh B gene in roots,leaves and tubers increased.This indicates that ABA and GA₃ treatment of potato seedlings can induce the expression of St RbohA and St Rboh B genes in different vegetative organs to varying degrees.3.The transient expression vectors including p Earlygate-St RbohA and p Earlygate-St Rboh B were constructed and transformed into tobacco leaves by Agrobacterium infection method.Under the laser confocal microscope,StrbohA and Strboh B were observed to function on the plasma membrane.4.From the upstream of the St RbohA and St Rboh B start codons,the promoter fragments St RbohAp and St Rboh Bp were cloned to the length of 1943 bp and 2032 bp,respectively.The expression vectors p BI121-St RbohAp and p BI121-St Rboh Bp were constructed and the promoter expression vector was transiently and stably transformed to tobacco seedlings and leaves.With the method of GUS histochemical staining,we found that both St RbohAp and St Rboh Bp have promoter activity and their ability to drive downstream GUS expression is weaker than the Ca MV 35S promoter.Among the stably transformed tobacco plants,we observed that the roots,stems and leaves of p BI121-St Rboh Bp transgenic tobacco plants showed obvious blue color;while in p BI121-St RbohAp transgenic tobacco plants,only in roots and leaves A small amount of blue dots appeared.This indicates that St RbohAp is a tissue-specific expression promoter,while St Rboh Bp constitutively expresses a promoter.Tobacco seedlings with transient transformation promoter expression vector were treated with 50μM ABA and GA₃ hormones for 3 days,and the expression of GUS gene was detected by qRT-PCR.The results showed that St RbohAp and St Rboh Bp can drive downstream GUS gene expression in response to ABA and GA signal,respectively.In summary,StrbohA and Strboh B are membrane proteins,and St RbohA and St Rboh B have higher expression levels in leaf and stem tissues.ABA and GA₃ treatment can induce their gene expression to show different changes in different vegetative organs.In this study,the dormancy mechanism of potato tubers was explored and we get some transgenic potato plant of over-expression and RNAi erpression of St RbohA and St Rboh B genes.The above results have important reference value for variety selection of the potato with different dormancy traits,agricultural production and storage processing.