Optimization Of Genetic Transformation System And Cloning And Function Of PgICE1

Pomegranate is an excellent fruit tree that combines economic benefits,ornamental value and medicinal value.There are many varieties of pomegranate,while there are some problems and defects in the varieties promoted on the market,among which prawns are frozen.The problem is more serious and has become a bottleneck in the cultivation of pomegranate.Therefore,the optimization of varieties has become a key issue in the production of pomegranate.The use of traditional breeding methods has the advantages of long breeding cycle and genetically high heterozygosity,which leads to slow breeding process,The application of transgenic technology in plant breeding has become a rapid and direct way to achieve the improvement of plant target traits.In order to establish a perfect pomegranate genetic transformation and transformation system,this study provides technical support for pomegranate genetic breeding.The cotyledons and leaves of pomegranate embryo culture seedlings are used as explants,and the genetic transformation system of pomegranate is optimized by Agrobacterium-mediated method.PgICE1,a cold-resistant gene related to pomegranate,was obtained,and bioinformatics analysis was carried out to construct an over-expression vector to transform Arabidopsis thaliana to verify the gene function,in order to lay a foundation for the genetic transformation of pomegranate cold-resistant new germplasm by genetic engineering.The main results are as follows:1.Pomegranate genetic transformation system optimization(1)Screening of optimum medium for cotyledon and leaf induced differentiation of buds after Agrobacterium infection.The infected explant material was connected to MS+0.3 mg·L-1 NAA+0.1 mg·L-1 KT+500 mg·L-1 Cef as control to screen the optimal TDZ concentration of induced differentiation buds..Cotyledon-inducing medium set the TDZ concentration to 0.5,1.0,1.5,2.0,2.5 mg·L-1,and the optimal medium for inducing cotyledon-derived shoot buds was:MS+2.0 mg·L-1 TDZ+0.3 mg·L-1 NAA+0.1 mg·L-1 KT+500 mg·L-1 Cef,the bud differentiation rate can reach 86.67%;the leaf induction medium TDZ concentration is set to 1.0,2.0,3.0,4.0,5.0 mg·L-1,the optimal medium for screening for differentiation of leaf buds was:MS+3.0 mg·L-1 TDZ+0.3 mg·L-1 NAA+0.1 mg·L-1 KT+500 mg·L-1 Cef,the bud differentiation rate is 27.78;(2)Screening for the optimum medium for inducing shoot buds to extract adventitious buds.The explants with shoots were connected to MS as the basic medium,and the medium with different concentrations of 6-BA and NAA was set up.The optimum medium for screening was:MS+1.5 mg·L-1 6-BA+0.5 mg·L-1 NAA+1.0 mg·-1 KT+0.3 mg·-1 GA3+500 mg·-1 Cef,the overall growth of adventitious buds is uniform,the growth was good,the adventitious buds were green and the leaves were stretched.;(3)Screening for the optimal screening medium.After the adventitious buds were stretched to 3-4 cm,they were cut from the base of the stem and MS was used as the basic medium.The proliferation medium with different concentrations of 6-BA and NAA was set to explore the optimal screening medium.The genetic transformation rate of adventitious buds induced by cotyledons was 45.45-51.11,the genetic conversion rate of adventitious buds induced by leaves was 33.33-38.89,and the optimal screening adventitious bud medium was:MS+mg·L-1 6-BA+0.3 mg·L-1 NAA+0.3 mg·L-1 GA3+200 mg·L-1 Cef,the proliferation coefficient of adventitious buds can reach 3.2,the growth buds grow uniformly,and the proliferation was strong;(4)Screening pomegranate tube grafting and transplanting optimal medium and carrying out graft regeneration of resistant adventitious buds.The Tunisia soft seed’ pomegranate seedlings were grafted on the cotyledon rootstocks of the ’Yuda seed’ seedlings,and the pre-experiment of the effects of different concentrations of 6-BA on pomegranate grafting and transplanting survival was explored.The medium with MS+0.1 mg·L-1 NAA as control and the concentration of 6-BA was set to 0.5,1.0,1.5 and 2.0 mg·L-1.The results showed that the optimum pomegranate was transformed into adventitious bud tube grafting.The medium was:MS+1.5 mg·L-1 6-BA+0.1 mg·L-1 NAA+200 mg·L-1 Cef,and the grafting survival rate and transplant survival rate were 96.67%and 70.00%,respectively;The adventitious buds were grafted in vitro,and 5 transplanted resistant bud grafted seedlings were obtained.The overall growth of the grafted seedlings was relatively weaker.2.Gene cloning and functional analysis of PgICE1(1)PgICE1 gene cloning and bioinformatics analysis.The open reading frame of PgICE1 gene was 1095 bp,and the predicted encoding was 394 amino acids,which was a hydrophilic acidic protein.It was predicted that PgICE1 was localized in the nucleus.In addition,the secondary structure analysis showed that α-helix,β-sheet was present in PgICE1.Four secondary structures of random coiling and elongation chain;predicting the tertiary structure of PgICE1 protein indicates that PgICE1 protein belongs to MYC2 type transcription factor protein;domain analysis revealed that PgICE1 gene has one transcription in multiple domains The bHLH domain of the factor is very similar to the functional domain of the cold-resistant gene ICE1 in most species;the phylogenetic tree analysis shows that the PgICE1 protein has the highest homology with the Eucalyptus camaldulensis ICE1,up to 73;the homology with the Vitis amurensis ICE1 can reach 60;(2)PgICE1 gene expression analysis.After low temperature treatment of pomegranate leaves,RT-PCR analysis showed that PgICE1 gene expression was rapidly induced when pomegranate was exposed to low temperature stress.(3)The PgICE1-pSAK277 overexpression vector was constructed and transferred into pomegranate and wild-type Arabidopsis thaliana.The resistant buds of PgICE1 gene were successfully obtained in pomegranate,and overexpression PgICE1 Arabidopsis plants were successfully obtained and subsequent research was in progress.The transgenic plants were resistant to cold.The sex test is in progress.