The Preliminary Analysis Of The Fuctions Of CmPYL4 And CmPYR1 Genes In Melon During Fruit Ripening

Melon is an important horticultural fruit crop,widely cultivated in the world.Because of its rich nutrition and excellent taste,it is very popular among consumers,so it is of great significance to study the ripening of melon fruit.At present,studies on abscisic acid receptors’ involvement in fruit ripening mainly focus on tomato and strawberry,but there are few studies on other fleshy fruits.By studying the functions of CmPYL4 and CmPYR1 genes during fruit ripening,we can enrich the research results in this field and provide a new direction for fruit ripening improvement.In this paper,the effects of ABA receptor genes CmPYL4 and CmPYR1 on melon fruit ripening were investigated using melon variety Hetao honeydew as research materials.The main results are as follows:(1)Melon abscisic acid receptor gene family members were identified,and a total of 12 members were obtained;Proteins encoded by family members possess hydrophobic ligand binding domains.35 completely conserved amino acid sites were obtained by multi-sequence alignment.Phylogenetic tree analysis can divide the family into three subfamilies.Expression calorimetry showed that CmPYL4 and CmPYR1 genes were highly expressed in fruits.(2)Analysis of gene expression patterns showed that CmPYL4 gene was highly expressed in fruits and seeds except for a small amount of CmPYL4 gene in roots.From 38 days to 44 days after pollination,the expression level of CmPYL4 gene in fruit seeds gradually decreased,but the expression level in fruit flesh gradually increased.CmPYR1 gene was expressed in all tissues,and higher expression level was found in roots,bisexual flowers,male flowers,and fruit pulp at 40 and 42 days after pollination.From 38 to 44 days after pollination,the expression level of CmPYR1 gene in seeds decreased gradually.(3)cDNA of CmPYL4 and CmPYR1 genes were cloned using total RNA from fruit pulp of mature melon as template,and the c DNA was ligated with overexpression vector p PZP221.Meanwhile,suitable double targets were designed to construct gene editing vectors of CmPYL4 and CmPYR1.In addition,subcellular localization vectors of CmPYL4 and CmPYR1 genes were constructed and expressed in tobacco,indicating that CmPYL4 and CmPYR1 proteins were located in cell membrane.(4)Melon was transformed by ovary injection,and by self-crossing and addition,T3 generation of CmPYL4 and CmPYR1 overexpressed transgenic homozygous lines were obtained.Compared with the 40.1 day maturity stage of wild-type melon,the target gene expression levels of T3 CmPYL4 overexpressed homozygous lines PYL4-OE21,PYL4-OE79 and PYL4-OE125 were 3.6,3.9 and 3.1 times higher than those of wild-type melon,and the maturity stage was 37,37 and 37.2 days,respectively.The expression levels of target genes of T3 CmPYR1 overexpressed transgenic homologous lines PYR1-OE6,PYR1-OE56 and PYR1-OE88 were 9.5,9.7and 10.5 times higher than those of wild type,respectively,and the maturity period was 35,35 and 35.1 days,respectively.It was significantly smaller than the ripening stage of wild type control fruit.(5)Yeast two-hybrid experiment of CmPYL4 and CmPYR1 genes was conducted.Both CmPYL4 and CmPYR1 proteins interacted with MELO3C013082.2.1 protein.CmPYR1 protein also interacted with MELO3C015408.2.1 protein at the concentration of 50 μmol/L abscisic acid.(6)In situ hybridization showed that CmPYL4 gene was expressed in ovule and CmPYR1 gene was expressed in pollen.At present,studies on abscisic acid receptors’ involvement in fruit ripening are mainly focused on tomato and strawberry,and there is almost no relevant research on other fleshy fruits.By studying the functions of CmPYL4 and CmPYR1 genes during fruit ripening,we can enrich the research results in this field and provide a new direction for fruit ripening improvement.