Functional Study Of Chitinase-like Gene PaCTL1 In Petunia

Flowers,fruits and seeds are the main reproductive organs of angiosperms,which occupy an important position in plant morphogenesis.Research on reproductive organs has always attracted much attention,especially in the field of flowering ornamental plant breeding.However,so far,most of studies focused on the color and aroma of flower,the molecular mechanism of flower morphology and fruit development has not been thoroughly studied and characterized.Previous studies have shown that plant chitinase plays an important role in plant resistance and vegetative growth,but there are still few reports on its regulation of reproductive organ development.In the previous study on the expression characteristics of petunia chitinase gene family members,our research group screened a chitinase-like gene PaCTL1（Gene ID:peaxi162scf00286g100222.1）which was preferentially expressed in the development process of flower organ and fruit.During this paper,the biological function of ornamental plants in the process of reproductive organ development was studied.The results will provide experimental evidence for dissecting the molecular mechanism of chitinase-like genes in the reproductive organ development of ornamental plants.The main experimental results are as follows:1.PaCTL1 was cloned from petunia.Sequence analysis showed that it was homologous with At CTL1（At1g05850）of arabidopsis,and their sequence similarity was 67.48%.The total length of its ORF sequence is960 bp,encoding 319 amino acids.The predicted molecular formula of PaCTL1 was C₁₅₈₉H₂₃₉₇N₄₁₁O₄₆₁S₂₃,and its molecular weight was 35.371k Da.The content of alanine and glycine is the highest,accounting for9.1%,The isoelectric point PI was 6.58.It is a hydrophilic protein with25 phosphorylation sites and chitinase＿GH19 domain belongs to Lyz-like super family.The amino acid sequence has a signal peptide,which is located in the region of 1-19 amino acids,with the shear site located between 19-20 amino acids.There wss no transmembrane structure,and the secondary structure is mainly irregular curl.The amino acid sequence of PaCTL1 was 92.54%identical to that of Nicotiana tomentosiformis.It was closest relative to Nicotiana tomentosiformis,Capsicum baccatum,Solanum lycopersicum and Solanum pennellii,which were members of the Solanaceae family.2.The analysis of expression characteristics showed that PaCTL1was preferentially expressed in flower buds,open flowers and tender capsules,though the expression level could also slightly be found in the roots,stems,leaves,flower stems of petunia.Furhermore,during the development progress of flowers from initial flowering to full flowering,the expression level of PaCTL1 in petals and pistils was gradually decreasing.Further experiments showed that the expression level of PaCTL1 in pistil decreased gradually after pollination,while the expression level increased gradually along with the capsule development.The promoter fusion expression vector p GWB433-pro PaCTL1 was constructed and transformed to the arabidopsis by agrobacterium-mediated genetic transformation.The histochemical analysis revealed that PaCTL1 was expressed in whole seedlings at the seedling stage,as well as in leaves at the adult stage,in flower buds,open flowers,and r siliques.The results of subcellular localization experiment show that PaCTL1 can be located on the cell wall.3.The suppression expression vector p RNAi-GG-PaCTL1 was constructed,and genetically transformed to petunia was by the leaf disc method.A total of three transgenic positive lines at the T₀generation were obtained.The morphological observation showed that,in comparison with the wild type,the flower organs of Line 2 and Line 11 had obvious changes,mainly including the length increase of corolla tube,sepal,corolla diameter,and style,as well as enlarged anther,enlarged stigma,enlarged ovary,uncurved sepals,wavy margins of petals and the abnormally prominent veins of petals.The pollen viability of Line 2 and Line 11 decreased by 27%and 44%respectively.In addition,the mature pods of Line 2 became smaller,the seed numbers in a single pod decreased by 90%,and the seeds became larger,the thousand kernel weight increased by 0.06 g.The Line 11 failed to set fruit normally.The overexpression vector p GWB520-PaCTL1 was constructed and genetically transformed to petunia by the leaf disc method.A total of two transgenic positive lines at the T₀generation were obtained.4.The overexpression vector p B7YWG2.0-PaCTL1 was constructed and transformed into wild-type arabidopsis by floral dipping method.The T₃generation plants were obtained through resistance screening.Phenotypic analysis of the mutant showed that compared with the wild type,the bolting time of the mutant was delayed by an average of 3 days,the flowering time was delayed by an average of 5 days,the average area of rosette leaves increased by 0.79 cm²on the 37th day,the plant height was 7.6 cm lower and the diameter of main flower stem increased by 0.21mm on the 49th day,the mature seeds became larger and lighter,and other phenotypes did not change significantly.5.Prokaryotic expression vector p GEX-4T-1-PaCTL1 was constructed,and transformed into E.coli JM109 competent state and E.coli Rosetta competent cells to induce the expression of PaCTL1 protein.The target protein was found to exist in the precipitation.It indicates that it may exist in the form of inclusion body.