Preliminary Analysis Of The Identification And Function Of Watermelon GAPDH Gene Family

Watermelon is an important economic crop in China,and its cultivation area and production rank first in the world.In recent years,with the expansion of facility cultivation area,breeding high-resistance,broad-adapted,and high-quality watermelon varieties has become one of the problems that needs to be solved urgently.At present,whole genome sequencing of watermelon has been completed,but subsequent research on gene function is relatively backward,especially for watermelon stress-related gene function research remains unclear.In addition,the genetic background of watermelon is relatively narrow.Therefore,the study and utilization of resistance-related genes can not only guide the breeding of new stress-tolerant watermelon varieties,but also provide a theoretical basis for the analysis of resistance mechanisms.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)is a constitutively expressed protein in the process of intracellular glucose metabolism.It is involved in the glycolysis pathway in cells and is closely related to the synthesis of ATP.Generally,GAPDH is abundant in the body and it is usually regarded as an internal reference protein.In this paper,the GAPDH gene family members of watermelon,melon,cucumber and pumpkin,which are the main crops of Cucurbitaceae,were screened and identified,and the family evolutionary relationships between different species were compared.At the same time,the non-sugars of the GAPDH gene family in abiotic stress and biological stress were also analyzed.The glycolysis function was preliminary verified,and the adversarial response genes Cla GAPDH3,Cla GAPDH4,and Cla GAPDH5 in the GAPDH family of watermelon were cloned.In order to further verify its function,an overexpression vector of GAPDH4 and a GFP vector were preliminarily constructed and transformed into Arabidopsis thaliana.T0 generation seeds.At present,the main research results of GAPDH gene function are as follows:(1)The number,phylogenetic relationships,gene structure,chromosome distribution,and collinearity of the GAPDH gene family members of watermelon,melon,cucumber,and pumpkin were determined.The GAPDH members of the four main cucurbit family watermelons,melons,cucumbers and squash were searched in the cucurbits crop database using the Arabidopsis model 8 At GAPDH gene protein sequences.They were named watermelon Cla GAPDH1-Cla GAPDH8,melon Cme GAPDH1.-Cme GAPDH7,Cucumber Csa GAPDH1-Csa GAPDH8,Pumpkin Cmo GAPDH1-Cmo GAPDH14.According to phylogenetic relationship analysis,the GAPDH gene family of watermelon,melon,cucumber and pumpkin can be divided into four subfamilies,which are Sub I,Sub II,Sub III,and Sub IV(having different functional domains: Gp_dh_N,Gp_dh_C,CP12,and Adledh,respectively).The analysis of its genetic structure revealed that there are certain differences in the number of introns contained in each member of different subfamilies,and the number of introns contained in the members of the same subfamily.They are basically the same,and their introns and exons are highly similar in organization.There are certain differences in the number of protein motifs contained in GAPDH gene family members in different subfamilies,and the species,number,and organization of conserved motifs in GAPDH gene family members in the same subfamily are very different.Strong consistency.Analysis Preliminary analysis of the identification and function of watermelon GAPDH gene family of chromosome distribution showed that the eight GAPDH genes in watermelon were mainly distributed on chromosomes Chr2,Chr3,Chr5,Chr9 and Chr11.The seven GAPDH genes in melon were mainly distributed on the six chromosomes Chr3,Chr5,Chr6,Chr7,Chr11 and Chr12.The eight GAPDH genes in cucumber were mainly distributed on chromosomes Chr1,Chr2,Chr3,Chr4,Chr6 and Chr7.There were 14 GAPDH genes in pumpkins distributed on 12 chromosomes.The GAPDH gene of Chr11 was most members found in cucumber.(2)In the present study,semi-quantitative q RT-PCR analysis of watermelon GAPDH gene was performed in different treatments,at different times and in different tissues.It was found that the expression of the GAPDH gene in watermelon was analyzed through different periods.The four genes of Cla GAPDH1,Cla GAPDH2,Cla GAPDH5 and Cla GAPDH6 showed significant up-regulation in watermelon leaves,Cla GAPDH6 and Cla GAPDH7 were up-regulated in flowers,and Cla GAPDH1 and Cla GAPDH2 were not significantly expressed in roots.Stress analysis showed that the expression of GAPDH gene in watermelon was different under stress conditions.Cla GAPDH3,Cla GAPDH4 and Cla GAPDH5 had a wide response under different treatments,while the expression abundance of other members was relatively low.This indicated that GAPDH gene could be induced by Na Cl,PEG,ABA,heat and cold stress.(3)Clone GAPDH3,Cla GAPDH4 and Cla GAPDH5 in watermelon GAPDH family were cloned;Cla GAPDH3,Cla GAPDH4 and Cla GAPDH5 gene sequences of watermelon were obtained by homologous cloning.After amplification by PCR,the target fragments were obtained,all located at about 1000 bp,and their size was basically consistent with that of the reference genome.The homology comparison analysis of the Sub III class Cla GAPDH3,Cla GAPDH4 and Cla GAPDH5 genes of watermelon revealed that they were highly similar.All of them contain Gp_dh_N domain which binds NAD(P)-N terminal and Gp_dh_C which is the C-terminal catalytic domain.Meanwhile,all watermelon GAPCs genes contain conserved serine residues.Pfam analysis showed that the 154 site serine has oxidative modification properties.