Establishment Of Gene Function Analysis System And Analysis Of AvFLS Gene Family In Apocynum Venetum

Apocynum venetum is a kind of drought-resistant and salt-tolerant desert plant which has great economic and medicinal value.Apocynum venetum can be used as a whole herb medicine,and the main medicinal components are flavonoids and cardiac glycosides.However,the regulation mechanism of the synthesis of secondary metabolites such as flavonoids and cardiac glycosides of Apocynum venetum is not clear.In order to analyze the biosynthesis regulation mechanism,a mature and convenient gene function analysis system is the prerequisite.Therefore,Apocynum venetum gene function analysis system is established from the aspects of screening reference gene,establishment and optimization of callus regeneration system and genetic transformation system.Based on the established gene function analysis system,the function of Flavonol Synthase(FLS)gene family in flavonoid synthesis can be obtained.The main results are as follows:1.Screening and verification of reference genesTUA,TUB,ACT,CYP,EF-1α,PPP2R2,PPP2R3,PPP2R5 and PGK were selected to determine the optimal reference genes in leaf,stem,and root of Apocynum venetum.The results showed that the two most stable reference genes in leaf,stem,root,and whole plantlet were CYP and TUA,PGK and PPP2R3,TUA and EF-1α,and CYP and PPP2R3,respectively.The above selected reference genes were cloned by PCR.2.Establishment and optimization of Apocynum venetum regeneration systemApocynum venetum roots,stems,leaves,and hairy roots were inoculated into MS medium supplemented with 0.5～4mg/L 6-BA,0.1～0.6mg/L NAA and 0.2～0.5mg/L 2,4-D for callus induction and regeneration plant examination.The results indicated that the optimal medium for the induction and adventitious bud differentiation of root calli was MS+0.5mg/L 6-BA+0.1 mg/L NAA+30g/L sucrose+6g/L agar(B4),and that for the induction and adventitious bud differentiation of stem and leaf calli was MS+1 mg/L 6-BA+0.2mg/L NAA+30g/L sucrose+6g/L agar(E4).The highest indeterminate adventitious bud induction rates were 68.33%,58.33%and 65.00%in roots,stems and leaves,respectively.And the mean number of adventitious bud were 5.02,1.34,and 2.31,respectively.On the basis of this result,a complete callus regeneration system was established using the root segments as explants,with a 100%rooting rate and 93%acclimation transplanting survival rate.3.Establishment of a genetic transformation system for Apocynum venetumAgrobacterium rhizogenes C58C1 were used to infect the roots,epicotyls,hypocotyls and leaves of Apocynum venetum and analyzing the infection time,OD600 value,acetosyringone(AS)concentration and co-culture time of hairy roots.The results showed that the transformation rate of hairy roots of different explants was as follows:hypocotyls>leaves>epicotyls>roots,the infection time was 20 min>25 min>15 min,OD600 value was 0.6>0.8>0.4,acetosyringone concentration was 100μmol/L>200μmol/L>0,and the co-culture time was 3 days>4 days>2 days.Therefore,the best genetic transformation conditions of hairy roots of Apocynum venetum were as follows:C58C1 with OD600 value of 0.6 was infected with hypocotyls for 20 minutes,then co-culture on the medium containing 100 μmol/L acetosyringone for 3 days,and the transformation rate of hairy roots could reach 42.25%.4.Identification,cloning and expression pattern analysis of AvFLS family genes in Apocynum venetumBased on the transcriptome data of Apocynum venetum,two AvFLS1 and AvFLS2 genes were identified,and the two genes were closely related to coffee.The AvFLS1 and AvFLS2 genes were cloned by PCR and the length of AvFLS1 and AvFLS2 are 951 bp and 1008bp long,respectively.The expression pattern analysis showed that AvFLS1 and AvFLS2 had different expression trends in roots,stems and leaves.AvFLS1 had the highest expression level in roots,while AvFLS2 had the highest expression level in leaves.The response of AvFLS1 and AvFLS1 gene expression to cadmium stress was also different.In the 0～160μmol/L CdCl2 stress treatment,the expression of AvFLS1 increased with increasing CdCl2 concentration in the roots,followed by a decreasing trend in the stems and leaves,and the expression of AvFLS2 decreased in the roots,followed by a decreasing trend in the stems and leaves.5.The role of AvFLS family genes in Apocynum venetum in flavonoid synthesisThe overexpression and interference vectors of AvFLS1 and AvFLS2 genes were constructed and transferred into Agrobacterium rhizogenes C58C1 respectively.The overexpression and interference hairy roots of AvFLS1 and AvFLS2 genes were obtained by the above optimized genetic transformation conditions;the overexpression and interference vectors of AvFLS1 and AvFLS2 genes were transferred into Agrobacterium tumefaciens LBA4404 respectively,and the genetic transformation was mediated by Agrobacterium tumefaciens Transformation method.