Discovery Of Cold-resistant Genes In Peach Based On TMT Quantitative Proteomics Analysis Technology

Peach(Prunus persica(L.)Batsch)is originated in China and belongs to Rosaceae.Peach trees are widely distributed in China.Hebei Province is a big province of peach cultivation,which is located in the northern limit of peach cultivation.In recent years,freezing damage of peach trees is serious.Therefore,it is significant to explore candidate genes related to cold resistance of peach for cultivating cold-resistant peach varieties in the future.Cold resistant peach varieties ‘Hunchun’,‘Zaoxialu’,‘Ruiguang27’ and ‘21shiji’,‘Mafim’,‘Zhonghuashoutao’ were used as test material.The cold resistance related genes in six peach varieties were preliminarily screened by TMT quantitative proteomics analysis technology,and the screened differential expressed genes were cloned and analyzed by RT-PCR and RT-q PCR.The obtained research results are as follows :1.A total of 53295 specific peptides were obtained by mass spectrometry analysis of three groups of protein samples : ‘Hunchun’ and ‘Zhonghuashoutao’,‘Zaoxialu’ and‘Mafim’,‘Ruiguang27’ and ‘21shiji’,6970 proteins were selected.A total of 106 differential expressed proteins were screened in cold-resistant peach and non-coldresistant peach(the difference multiple ≥ 1.5,P≤ 0.05),which obtained 63 upregulated and 43 down-regulated.These differential expressed proteins were mainly located in chloroplast(19.30%),cytoplasm(15.79%),vacuole(10.53%),cell membrane(8.77%),cell wall(7.02%),nucleus(5.26%),mitochondria(5.26%),etc.GO analysis showed that these proteins were mainly involved in 18 biological processes,20 molecular functions and 3 cell components.Four genes related to cold resistance of peach Pp ATP9,Pp ATP4,Pp3 GT and Pp FAD6 were further identified by differential expression analysis.2.Cloning analysis found that the full-length of Pp3 GT and Pp FAD6 was 1434 bp and 1326 bp respectively.Cluster analysis showed that Pp3 GT protein was clustered with apple(Malus domestica)and almond(Prunus dulcis)3GT protein,Pp FAD6 protein was clustered with plum(Prunus mume)and monthly rose(Rosa chinensis).RT-q PCR results showed that Pp3 GT gene was induced to express in ‘Hunchun’ peach after low temperature treatment.RT-q PCR results showed that the relative expression of Pp3 GT gene and Pp FAD6 gene in ‘Hunchun’ peach increased primarily and then decreased after low temperature treatment,the expression of Pp3 GT gene in‘Zhonghuashoutao’ decreased,and the expression of Pp FAD6 gene in‘Zhonghuashoutao’ decreased primarily and then increased.3.Cloning analysis found that the full-length of Pp ATP9 and Pp ATP4 were 243 bp and 597 bp respectively.They are the unstable protein.The cluster analysis showed that Pp ATP9 was clustered with apple(Malus domestica)Md ATP9,Pp ATP4 was clustered with almond(Prunus dulcis.)Pd ATP4 and plum(Prunus cerasifera)Pc ATP4.RT-q PCR results showed that: in the natural wintering process the expression of Pp ATP9 gene in the buds of these three peach varieties was significantly correlated with temperature change trend,it increased primarily and then decreased in the phloem of ‘21shiji’ and ‘Zhonghuashoutao’,while it was persistently decreased in ‘Hunchun’.The expression of Pp ATP4 gene in buds of these three peach varieties showed no significant correlation.In phloem,it showed persistently decreased expression in‘21shiji’ and ‘Zhonghuashoutao’,while in ‘Hunchun’ peach,it increased primarily and then decreased.