Functional Analysis Of AtGPRP3 In The Growth Of Arabidopsis Seedlings

The GPRP?Glycine and proline-rich protein?protein family genes widely distributed in plants have been reported to play an important role in the growth and development of plants.However,there are few reports on the in-depth study of the function of GPRP protein genes in plants.In this study,the Arabidopsis AtGPRP3 gene,which has been reported and has not been studied for biological function,was used as the research object.Based on the analysis of the basic structure,protein evolutionary relationship and expression characteristics of the gene,the transgenic materials of overexpression of AtGPRP3,deletion expression and functional complementation was further created.And their effects on the growth and development of Arabidopsis seedlings were analyzed.It was found that AtGPRP3 gene affected the growth rate of Arabidopsis seedlings.The finding laid the foundation for further study of the biological function of AtGPRP3gene.The main results are as follows:1.The basic characteristics,protein evolutionary relationship and expression patterns of the AtGPRP3 gene in Arabidopsis thalianaBioinformatics analysis revealed that the AtGPRP3 gene contains two exons and one intron,encoding a total of 179 amino acids.The isoelectric point?pI?of the protein is 9.75 and the molecular weight?Mw?is 17.8 kDa.Protein evolution analysis showed that the GPRP protein family genes did not differentiate significantly in monocotyledonous and dicotyledonous.The results of GUS staining and qRT-PCR showed that the AtGPRP3 gene was mainly expressed in the leaves of Arabidopsis thaliana,and was less expressed in mature pods.Subcellular localization results indicate that AtGPRP3 protein is mainly expressed in the nucleus.2.The creation of transgenic Arabidopsis material of AtGPRP3 overexpression,deletion expression and functional complementationThe overexpression vector of AtGPRP3 was constructed with pCXSN plasmid.The deletion mutant of AtGPRP3 and its functional complementary transgenic genetic material were constructed with plasmid Yao.ATU6₃5s.HPT and pCAMBIA1300-Myc respectively.At present,three types of overexpression,deletion expression and functional complementation transgenic homozygous lines of AtGPRP3 gene was obtained,and they were used for subsequent functional analysis experiments.3.AtGPRP3 affects the growth and development of Arabidopsis seedlingsTransgenic Arabidopsis thaliana materials of wild-type Arabidopsis thaliana,AtGPRP3 gene overexpression,deletion expression and functionally complementary were cultured on two mediums of pH 5.8 and pH 8.0,respectively.The fresh weight of seedlings in various materials was counted.The results showed that compared with the fresh weight of wild-type Arabidopsis seedlings,the fresh weight of the shoots of AtGPRP3 overexpressing transgenic plants was significantly reduced,and the fresh weight of the above-ground seedlings of AtGPRP3 deletion-expressing transgenic plants was significantly increased,while there was no significant difference in the fresh weight and wild type of the functionally complementary transgenic plants.It indicated that AtGPRP3 may have a negative regulation effect on the fresh weight of Arabidopsis seedlings,which may affect the growth rate of Arabidopsis seedlings.4.Screening,identification and transgenic function verification of AtGPRP3interaction genesBased on the construction and preliminary screening of cDNA library of Arabidopsis thaliana seedlings,the yeast two-hybrid?Y₂H?and bimolecular fluorescence complementation?BiFC?were further identified,and three interaction genes?AtCAT2,AtCLC3 and AtCYSD1?of AtGPRP3 were obtained.Furthermore,the CRISPR-Cas9 technology was used to focus on the deletion of the Arabidopsis thaliana transgenic mutant of AtCAT2,and the growth was carried out on two mediums of pH 5.8 and pH 8.0,respectively.Statistical analysis showed that the fresh weight of the seedlings of AtCAT2 was significantly lighter than that of wild-type Arabidopsis seedlings.This result is similar to the phenotype of the AtGPRP3overexpressing transgenic line;thus,we preliminarily speculated that the combination of AtGPRP3 and AtCAT2 may have an inhibitory effect on AtCAT2 itself,but further confirmation is required.