Establishment Of Strawberry Transgenes System And Functional Analysis Of ABA Receptor PYR1 Gene

ABA(abscisic acid)as a plant hormone,has been confirmed not only plays an important role in the regulation of plant dormancy,aging process and anti-stress process,but also an important factor in regulating fruit development.In recent years,although the signal recognition on the ABA and its molecular mechanisms of signal transduction achieved a breakthrough in the pattern plant,the molecular mechanism of ABA regulation of fruit development is unclear.In this study,strawberry as tested material,based on the establishment of stable genetic transformation system in strawberry and establishment of virus induced gene silencing system in strawberry fruit,analysis the role of abscisic acid receptor gene PYR1 in fruit ripening,in order to reveal the ABA regulation of fruit development signal recognition mechanism.(1)Strawberry PYR1 genes firstly cloned.The gene is 627bp length,encoding 208 amino acids,named FaPYR1,and gene accession number is:JF268669.1.The cloned nucleotide sequence of FaPYR1 cDNA inserted reversely into restriction site Bgl? and Spe ? of plasmid pCAMBIA1304,constructing the antisense plant expression vector pCAMBIA1304-FaPYR1;424bp of FaPYR1 sequence was choosed and inserted into the restriction site Xba ? and Kpn ? of TRV2 viral vector,constructing a virus-mediated gene silencing vector pTRV2-FaPYR1,(2)Using Agrobacterium-mediated gene silencing system,the virus vector carrying FaPYR1 gene was injected into the green fruit two weeks after flowers,and observed after oneweek.The results showed that FaPYR1 gene silencing can make strawberry fruit unripening,the silence site of the strawberry fruit was maintained white,and can not color,and other site becomed red.While the control strawberry fruit becomed red and softening.FaPYR1 gene expression was reduced using semi-quantitative RT-PCR,quantitative RT-PCR,Western Blot test.Gene expression of its downstream signaling has been reduced to varying degrees with quantitative RT-PCR method.These results show that,F?PYR1 gene plays an important role in strawberry fruit development.This study also found that injection in strawberry fruit stalk is an effective method to establish virus induced gene silencing.(3)Leaves of 'hongyan' strawberry as explants,FaPYRl gene as reporter gene,Agrobacterium-mediated genetic transformation system was established with 'hongyan' strawberry:bacterial concentration was OD600= 0.4;infection time was 8min;200mg/L of Cb was antibacterial antibiotics,and 20mg/L of Kan was a screening antibiotics;co-cultured for 2 days;co-culture medium:MS + TDZ(2.0mg/L)+ IBA(0.2mg/L);selection medium:MS + TDZ(2.0mg/L)+ IBA(0.2mg/L)+ Kan(20mg/L)+ Cb(200mg/L);elongation medium:MS + 6-BA(0.5mg/L)+ NAA(0.1 mg/L)+Kan(20mg/L)+ Cb(200mg/L).