| Environmental stress is the main reason affecting peach growth and geographical distribution,which is mainly manifested as low temperature stress in northern China.The winter climate in Hebei Province is very cold and lasting for a long time.Both the growth and development of peach leaves in early spring and the nutrient accumulation of leaves in late autumn are greatly affected by low temperature stress.Therefore,the research on low temperature adaptability of peach leaves has gradually become one of the problems to be clarified in the breeding process of peach trees in the north.RNA-seq was performed on ‘ Hunchun ’ peach leaves at different stages under normal temperature and low temperature stress.Sequencing results showed that two RBD family genes PpRBD1 and PpRBD2 were closely related to low temperature stress.Based on the above results,this study verified the function of this gene in transgenic Arabidopsis and tobacco by constructing PpRBD1 transgenic vector.In addition,a new RBD family gene,PpRBD2,was identified from peach in this study,which also had a RRM domain,and the expression level of this gene under low temperature stress was preliminarily analyzed.The results show that:1.The transcriptome sequencing analysis of ‘ Hunchun ’ peach leaves under normal temperature and low temperature stress was carried out.A total of 57.7Gb high quality Clean Data were obtained.The total number of sequencing data after quality control of each sample was between 40.93 ～ 47.01 million,the percentage of Q20 base was more than 98%,and the percentage of Q30 base was more than 93.87%;The sequences were aligned with the reference genome of peach,and the proportion of Clean reads that could be aligned to the genome of each sample was between 92.80%and 95.91%.There were 4000 up-regulated genes and 5121 down-regulated genes in peach leaves after 17 days of low temperature treatment.There were 4224 up-regulated genes and 5096 down-regulated genes in peach leaves after 30 days of low temperature treatment.A total of 22 RBD family genes located in chloroplast were found in the sequencing data.Based on the previous research results and the differential expression analysis data of this experiment,two candidate genes PpRBD1 and PpRBD2 involved in low temperature stress resistance of peach were found.2.The function of PpRBD1 gene in response to low temperature stress was verified in Arabidopsis and tobacco.The transgenic results showed that under 4°C low temperature stress,both PpRBD1 replenishable Arabidopsis plants and wild-type plants could grow slowly.PpRBD1 overexpression Arabidopsis plants can grow normally,while Arabidopsis rbd1-1 mutant plants grow slowly and new leaves appear yellowing phenomenon.In PpRBD1 transgenic tobacco,with the extension of low temperature stress time,the wild type appeared wilting phenomenon after 23 days,and the leaves fell off and died after 30 days.The transgenic plants can grow normally after 23 days,and some leaves wilted after 30 days.The above results showed that PpRBD1 gene could improve the resistance of Arabidopsis and tobacco to low temperature stress.3.A new RBD family gene named PpRBD2 was cloned from peach for the first time.The full length of the gene is 2401 bp and the coding region is 897 bp,encoding298 amino acids with two RRM domains.The gene is highly homologous to RBD genes in Prunus avium,Malus domestica and Pyrus × bretschneideri.RT-q PCR results showed that after low temperature treatment,the relative expression levels of PpRBD2 gene in ‘ Hunchun ’,‘ Maotao 1-1 ’ and ‘ 21 Shiji ’ peach varieties showed a trend of first decreasing and then increasing.With the extension of low temperature treatment time,the expression levels of PpRBD2 gene in ‘ Hunchun ’ and ‘ Maotao 1-1 ’ peach varieties were significantly higher than those in ‘ 21 Shiji ’ and ‘ Zhonghuashoutao ’peach varieties,indicating that the gene was closely related to cold resistance of peach. |
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