Identification And Validation Of Atp6 Interaction Protein Of UG93A Male Eterility Related Gene In Kenaf

Kenaf(Hibiscus cannabinus L.)is a herbaceous non-woody fiber plant which is mainly grown in Asia and Africa.The bast fiber composed of 75%cellulose and 7%lignin and offer the advantage of being biodegradable.It has versatile applications i.e.paper making,animal bedding,building material,carpet backing,cordage,forage for livestock and biomass crop.the utilization of its heterosis is very get popularized in kenaf breeding.In 2002,professor zhou ruiyang has discovered the male sterile mutant of kendra in lingshui,hainan,and transformed it into male sterile line.In 2008,the world's first kenaf "three-line"hybrid was identified by experts organized by the national agricultural extension service center.The breeding of kenaf cytoplasmic male sterility(CMS)lines and the utilization of heterosis have been made great improvements.Previous studies of our research group showed that the mitochondrial gene atp6 was closely related to kenma CMS and atp6.Meanwhile,the CDS sequence of kenma atp6 was used to construct the yeast bait vector.The kenma transcription factors HcMYC2a and HcMYC2b were screened through the yeast two-hybrid library to interact with atp6,respectively.In order to verify the functional areas of interaction of ATP6 with HcMYC2a and HcMYC2b protein,the infusion DNA fusion method was used in this study.The yeast bait vector was constructed using a short ATP6 sequence and the yeast prey vector was constructed by short HcMYC2a and HcMYC2b sequences.The core functional region of interaction between ATP6 and HcMYC2a and HcMYC2b of kenma were verified by yeast top hybridization and bimolecule fluorescence complementary technique,respectively.The main findings are listed below:(1)the cDNA of erythum UG93A and UG93B was used as a template for the Infusion DNA for the construction of short-cut yeast bait recombinant vectors,namely PGADT7-catp6a-1,PGBKT7-atp6a-2,PGBKT7-atp6b-1 and PGADT7-atp6b-2.The recombinant vector PGADT7-HCMYC2a-1,PGADT7-HCMYC2a-2,PGADT7-HCMYC2b-1 and PGADT1-HCMYC2b-2 were constructed.(2)Above mentioned yeast bait vector was transformed into Y2H and prey vector into Y187,respectively,and recombinant plasmids with different short fragments were tested by yeast double hybridization.Data showed that atp6A-2,HCMYC2b-2,atp6B-1,HCMYC2b-1,atp6B-2,HCMYC2a-1,atp6b-2 and HCMYC2b-1 hybridized on the medium of SD/-trp-leu-his-ade/AbA/x-alpha-gal,respectively.This indicate that these protein domains were interacted with each other.(3)Infusion DNA was used to construct the C-terminal bimolecule fluorescence complementary vector pSPYCE-atp6a,pSPYCE-atp6b,pSPYCE-HCMYC2aa,pSPYCE-HC.MYC2ab,pSPYCE-HCMYC2bb,and N-terminal bimolecule fluorescence complementary vector pSPYNE-atp6a,pSPYNE-atp6b,pSPYNE-HCMYC2aa,pSPYNE-HCMYC2ab,pSPYNE-HCMYC2bb.(4)Above recombinant vectors were injected into the tobacco leaves of b.bunge in different combinations.The data showed that tobacco leaves injected with ATP6A,HCMyc2bb,ATP6B,HCMyc2aa,ATP6B,HCMyc2ab,ATP6B and HCMyc2bb all emit yellow fluorescence signals.which indicating that these proteins were interacted in tobacco leaves.