Recombination Of DPBF2,DPBF3,DPBF4,DPBF5 And At5G42910 Genes Of ABI5 Subfamily And Construction Of Overexpressed Transgenic Arabidopsis

The ABI5(ABA insensitive 5)subfamily participates in the regulation of plant growth and development.In the absence of ABA,transgenic plants overexpressing full-length ABI5 subfamily members often have insufficient traits due to the existence of transcriptional activity inhibitory regions.Therefore,ABI5 subfamily members are often used as negative regulators or overexpressing plants for downstream genes.Inconsistent reports of phenotypic changes.ABI5 subfamily transcription factors are highly conserved in amino acid sequence.According to their conservation and previous studies,it has been shown that the conserved region II(CRII)of the 9 members of the ABI5 subfamily has transcriptional activation activity,and DPBF4 CRII has the strongest transcriptional activation activity,ABI5 The conserved phosphorylation site has a significant inhibitory effect on its transcriptional activation activity.Based on this,we removed the transcription inhibitory region of each member,selected DPBF4 CRII and the DNA binding domain(bZIP)of other members to construct an active form of recombinant transcription factor,and then selected transgenic plants with stable genetic phenotype after overexpression in Arabidopsis.Omics sequencing analysis of each overexpression plant to understand the growth phenotypes and molecular mechanisms regulated by the 9 members of the ABI5 subfamily during plant growth and development,and to clarify the detailed biological functions of each member.The main results of our rearch are as follows:(1)Construction the recombinantvectors: p1304-4IID2 bZIP,p1304-4IID3 bZIP,p1304-4IID4 bZIP,p1304-4IID5 bZIP and p1304-4II910 bZIP.(2)The p1304-4IID2 bZIP,p1304-4IID3 bZIP,p1304-4IID4 bZIP,p1304-4IID5 bZIP and p1304-4II910 bZIP recombinant vectors were transformed wild-type Arabidopsisby Agrobacterium GV3101.We obtained overexpressing transgenic plants six lines of A.thaliana 910 and eight lines of A.thaliana D3.Currently,we are also being screened and passed down to the T1 generation(the T2 generation is being screened).Both A.thaliana D2 and A.thaliana D4 had screened 10 transgenic lines,the T1 generation is being screened and passed down.(3)Construction the yeast two-hybrid vector AD/BD-ABF1 bZIP,AD/BD-ABF2 bZIP,AD/BD-ABF3 bZIP,AD/BD-ABF4 bZIP,AD/BD-ABI5 bZIP,AD/BD-DBBF2 bZIP,AD/BD-DBBF3 bZIP,AD/BD-DBBF4 bZIP and AD/BD-At5G42910 bZIP.(4)Through the yeast two-hybrid system,all the homo/heterodimer patterns that can be formed by 9 members of the ABI5 subfamily are analyzed.The results show that only ABF3 and ABI5 can form homodimers among the 9 members;ABI5 can form heterodimers with ABF2 and ABF4,ABF1 and ABI5 can also form weak heterodimers.(5)The Arabidopsis transgenic line that overexpressing recombinant ABI5 was named A.thaliana MX-1-1 and overexpressing ABI5 full-lengthgenic linewas named A.thaliana MX-4-4.those were tested by q RT-PCR for the expression level of the respective phenotype-related genes at the transcription level.In the ABA signaling pathway,ABI5 regulates the transcriptional level of its downstream genes,such as drought response genes RD29 A and RD29 B,late embryogenesis genes At EM1 and At EM6,flowering suppressor gene FLC,and regulation of seed size-related gene SHB1.The results showed that as the seedlings grew,compared with the wild type,the m RNA expression of ABI5 and RD29A were significantly up-regulated in A.thaliana MX-1-1,and increased by 246 and 1.8 folds respectively in the 24-day-old seedlings,while in A.thaliana MX-4-4,ABI5 was first increased by 47 folds in14 days old seedlings,and decreased to 0.2 folds in24-day-old seedlings with the growth and development of plants.RD29 Adecrease to 0.6folds in 24-day-old seedlings of A.thaliana MX-4-4 compared with wide type;the expression of FLC,SHB1,RD29 B,At EM1 and At EM6 in A.thaliana MX-1-1 and A.thaliana MX-4-4 first increased and then decreased with the growth and development of seedlings:the m RNA expression of FLC in 14-day-old Arabidopsis seedlings of A.thaliana MX-1-1 and A.thaliana MX-4-4 increased by about 6-fold and10-foldrespectively,in 24-day-old Arabidopsis seedlings of A.thaliana MX-1-1 and A.thaliana MX-4-4,it dropped to 0.3 of that of the wild type.The m RNA expression of SHB1 increased by 2.5 folds in 14-day-old A.thaliana MX-1-1,and the expression in24-day-old seedlings was not significantly different from that wild-type.At 14-day-old seedlings of A.thaliana MX-4-4 increased by 3.5 folds and decreased to 0.4 folds of24-day-old seedlings;m RNA expression of RD29 B in A.thaliana MX-1-1 did not change significantly,in 24-day-old seedlings of A.thaliana MX-4-4 decreased to 0.6folds.Meanwhile,the expression levels of At EM1 and At EM6 were both up-regulated about 5 folds in 14-day-old seedlings of A.thaliana MX-1-1,and then they were down-regulated to no significant difference in 24-day-old seedlings.In 14-day-old seedlingsof A.thaliana MX-4-4,they were up-regulated about 9 folds and 11 folds,then,they were all down-regulated to 0.2 folds of 24-day-old seedlingscompared with wild-type.The above results indicate that the down-regulation of these related genes are more significantin A.thaliana MX-4-4.(6)The proteomics results of A.thaliana MX-1-1 show that compared with the wild type,a total of 35 differential proteins have been detected in A.thaliana MX-1-1 overexpressing transgenic plants,which are involved in signal transduction,immune response,and energy metabolism.Active ABI5 regulates the growth and development of plants by participating in the biosynthesis and metabolism of ABA and the GA signal pathway.