Cloning And Functional Analysis Of BsGAI1 And BsGAI2 Genes Of DLLA Protein Family Of Populus Euphratica

Buxus sinica var.parvifolia has very high ornamental value,with vigorous posture and dwarf type,but few studies have been reported on its dwarfing mechanism.GA signaling transduction pathway or biosynthesis,which is impeded,is an important factor in plant dwarfing.DELLA protein is an important negative regulator in GA signaling transduction pathway.In this paper,BsGAI1 and BsGAI2 genes of the DELLA protein family,which related to dwarf of Buxus sinica var.parvifolia,were studied.The results are as follows:(1)DELLA protein gene,named BsGAI1,BsGAI2 were cloned from leaves of Buxus sinica var.parvifolia using homological cloning and RACE(rapid-amplification of cDNA ends),the complete cDNA sequences were obtained: 2576 bp,2305bp,and the coding region length was1884 bp and 1836 bp,respectively,encoding 627 aa and 612 aa amino acid sequences.The physical and chemical properties of BsGAI1,BsGAI2 genes were analyzed.The molecular formula of BsGAI1 was C2951H4637N821O928S29 and the molecular weight was 67.39 kD.The molecular formula of BsGAI2 was C2891H4541N811O908S30 and the molecular weight was 66.15 kD,both proteins are unstable hydrophilic proteins.The secondary structure of BsGAI1,BsGAI2 gene was mainly ?-helix and random curl.BsGAI1,BsGAI2 encoded protein has a functional active site and a conserved unit of the DELLA gene family.There are much conserved DELLA and TVHYNP domains at the N-terminus.The C-terminal structure is highly conserved and exists VHIID,RVER and SAW domains.(2)Real-time quantitative analysis of BsGAI1 and BsGAI2 genes in different tissues of Buxus sinica var.parvifolia.The results showed that the expression of BsGAI1 gene was the highest in stem and the lowest in the root,the expression of BsGAI2 gene was the highest in stem and the lowest in flower.The differences in the expression of BsGAI1 and BsGAI2 genes in different stages of Buxus sinica var.parvifolia were analyzed.It was found that the expression of BsGAI1 in leaves,stems and roots in July of 2016 was higher than that in January and September,which indicated that Buxus sinica var.parvifolia grew stronger in the summer,the expression of BsGAI1 would increase.In September,the expression of BsGAI1 in the stem was significantly higher than that in the leaf and root.The expression of BsGAI1 in the stem increased with the passage of time,these results all indicate that BsGAI1 gene may play an important role in the process of shorting stem internode for Buxus sinica var.parvifolia dwarf.Compared with BsGAI1,the expression of BsGAI2 in January,July and September was different.The expression level of BsGAI2 in these three months was not the highest in stem,which indicated that different genes expressed in different stages of plant have differences.Construction of GFP fusion expression vector of BsGAI2 gene by double digestion,and the fusion expression vector was transformed into the model plant tobacco by Agrobacterium tumefaciens.The differences of transgenic tobacco and wild tobacco were identified by genomic PCR,phenotypic analysis,GFP fluorescence detection,IAA and GA3 endogenous hormone determination and spraying exogenous GA3.Compared with wild-type tobacco,BsGAI2 transgenic tobacco showed slow growth,dwarfing trend,and the internodes were shorter and stout,slow root growth with less fibrous roots.Transgenic tobacco and wild-type tobacco tissues were observed by signal acquisition of GFP fluorescent protein using fluorescence microscopy.It was found that in the transgenic tobacco stem,observing that the xylem of the transformed seedlings had a bright fluorescent signal,and the cells in the intervening focus position could see a clear signal of fluorescence aggregation.Meanwhile the rhizome buds,leaves and roots also had bright fluorescent signals,wild-type of tobacco had no signs of fluorescence aggregation.IAA and GA3 endogenous hormones were found that the IAA endogenous hormone content of BsGAI2 transgenic tobacco and wild type tobacco was not significantly different,while the GA3 content of transgenic tobacco was higher than that of wild type,the increase of GA3 content did not change the state of plant dwarfing,indicating that BsGAI2 gene plays an important role in regulating plant dwarfing.After spraying exogenous GA3,transgenic plants and wild-type tobacco internode elongation and stem growth rate was significantly accelerated,which indicated that the BsGAI2 gene was transferred into the wild type tobacco,the expression level of the normal gene in the plant was inhibited,resulting in plant dwarfing.With the exogenous GA3 spraying,the GA expression in the plant was restored to normal,and the growth of the plant gradually returned to normal level.