Phenotype Of Arabidopsis Thaliana EXPA Multi-Gene Mutants Using CRISPR/Cas9 System And Transgenic Plants Overexpressing NcEXPA8 Gene Of Neolamarckia Cadamba

As one of the fast-growing tree species,Neolamarckia cadamba has been little studied about its fast-growing molecular mechanism.The growth of plant cells is restricted by the cell wall.Expansin(EXP)is a kind of cell wall growth regulator,which plays an important role in the process of cell wall modification and cell expansion and is closely related to the growth and development of plants.In this study,we constructed a phylogenetic tree of NcEXPA8 protein in N.cadamba and 26 EXPA proteins in Arabidopsis thaliana,and found that NcEXPA8 belongs to the same branch as the subfamily A of EXPA proteins in A.thaliana.Therefore,we selected the gene mutants of the members of subfamily A,At EXPA3,At EXPA4,At EXPA6,At EXPA9 and At EXPA16,to carry out experiments.It revealed that the functional redundancy of subfamily A genes and indicated that the NcEXPA8 gene has similar functions to the At EXPA subfamily A gene.Meanwhile,we performed seed germination experiments on N.cadamba and NcEXPA8 transgenic A.thaliana,suggesting that NcEXPA8 gene may be involved in the process of N.cadamba seed germination.Through the above research,we have created the A.thaliana EXPA multi-gene mutants and laid the foundation for the functional research of the gene NcEXPA8 in N.cadamba.The main results are listed as follows:1.We identified A.thaliana single-gene mutants atexpa3,atexpa4,atexpa6,atexpa9,and atexpa16 by three-primer method,and crossed two homozygous mutants of atexpa4 and atexpa16 to obtain atexpa4/16 double-gene mutation homozygote.However,compared with the wild type,there were no obvious phenotypic difference in single-gene mutants and double-gene mutant.2.Using CRISPR/Cas9 gene editing technology,we constructed an At EXPA multi-gene editing vector p HEE401E-At EXPA3/6/9,and transformed it into the double gene mutation homozygote atexpa4/16.The three-gene mutation homozygotes atexpa3/4/16,atexpa4/6/16,and four-gene mutation homozygotes atexpa3/4/6/16 were obtained by screening homozygous non-transgenic plants.Then,the p HEE401E-At EXPA9 vector was constructed to transform the four gene mutation homozygotes atexpa3/4/6/16 to obtain homozygous non-transgenic five-gene mutant homozygote atexpa3/4/6/9/16.3.Quantitative Real-time PCR(RT-q PCR)technology was used to analyze the expression levels of the three-gene mutants atexpa3/4/16,atexpa4/6/16,and the four-gene mutant atexpa3/4/6/16.The results showed that both in rosette leaves and inflorescence stems,the expression levels of the mutational genes of the corresponding mutants all decreased to varying degrees.4.We performed phenotypic observations on the multi-gene mutants atexpa3/4/16,atexpa4/6/16,and atexpa3/4/6/16.The results showed that the multi-gene mutants all showed significant growth inhibition,including that the rosette area became smaller,the number of leaves decreased,the plant dwarfed,and the inflorescence stem thickness became smaller,compared with the wild type.Meanwhile,the length of the hypocotyl became shorter but the number of longitudinal epidermal cells did not change significantly,indicating that the shortening of the hypocotyl is mainly caused by the shortening of the cells,but the growth inhibition of different mutants was not exactly the same.5.We analyzed the microstructure of the multi-gene mutants atexpa3/4/16,atexpa4/6/16,and atexpa3/4/6/16.The results showed that in the inflorescence stem cross-section structure of the multi-gene mutant,the cortex was significantly thinner and the cortical cells were significantly smaller,compared with the wild type.In the hypocotyl cross-section structure,the hypocotyl growth of multi-gene mutants was inhibited including the underdeveloped xylem and even growth defects.6.We overexpressed the N.cadamba gene NcEXPA8 in A.thaliana multi-gene mutants atexpa3/4/16,atexpa4/6/16,and atexpa3/4/6/16.The results showed that the rosette area of the three over-expressing lines was significantly larger than that of the original mutants and the leaf number increased.Meanwhile,the plant height also recovered to a certain extent,and some phenotypes returned to the wild type.7.In the N.cadamba seed germination experiment,the expression level of the gene NcEXPA8 was highest when the seed was just germinated.Subsequently,the radicle continued to develop,but the expression level was significantly reduced and maintained at a low level.In the germinated seeds of three NcEXPA8 transgenic A.thaliana lines,the gene NcEXPA8 had a high expression level,which was significantly higher than other endogenous genes specifically expressed in seed germination,but the overexpression of NcEXPA8 gene did not affect the expression of these genes.Moreover,the overexpression of NcEXPA8 gene also reduced the sensitivity of A.thaliana seeds to ABA and increased its sensitivity to GA.