Cloning And Primary Function Analsis Of Small Heat Protein Genes In Prunus Mume

Mei flower (prunus mume Sieb.et Zucc.) is native to China, greatly loved by the people. It is widely applied in urban greening. Current research about Mei flower concentrated on the germplasm resources and tradition breeding. However, researchers are paying little attention to the molecular breeding. It has proved that the small heat shock protein(sHSP) can improve the resistence of the plants, but the researches about regulatory mechanism that the sHSP responds to multi-stresses are few, and fewer ones about Mei flower.The sHSP genes of Prunus mume ’Xue Mei’ was cloned to analyse the genes structures’s differences, and the expression difference of PmsHSPs was detected after heat stress by Semi-quantitative RT-PCR. Then, a plant expression vector of the PmsHSPs was constructed, and was used for transformation of Arabidopsis thaliana. The function of PmsHSPs was analysed by observing the morphological differences among WT and transformated Arabidopsis thaliana during normal and stress condition. The main results are as follows:1. The expression analysis of PmsHSPs under heat stress condition. The subject was the branches with leaf buds of ’Xue Mei’. After two weeks of hydroponics, leaves stretched out and the branches were taken the treatment of different duration in the light incubator. The time arrangement was30min,1h,2h,3h,4h,12h and24h. Then the subject were then preserved in-70℃. After the analysis of semi-quantitative PCR, under nature condition, the expression of PmHSP17.6-CⅢ and PmHSP17.9-CⅡ were not found. Under1h treatment, the expression level of these two genes was the highest, yet the lowest point was under the24h treatment. Thereforem, we assumed that PmHSP17.6-CⅢ and PmHSP17.9-CⅡ were inducible expression genes. The expression of PmHSP18.0-CⅠ existed under nature condition and had no obvious changes under different treatment duration. As a result, we assumed that PmHSP18.0-CⅠ was a constitutive expression gene.2. The cloning of PmsHSP. According to the information of comparation of the Mei flower transcriptome sequencing, we chose11PmsHSPs genes. Among them,6 cytoplasm genes,2mitochondrial genes, a chloroplast gene, a endoplasmic reticulum gene and a peroxisome gene. The molecular weight of these sHSPs were from17.6KD to26.0KD. Using ordinary PCR and Tail-PCR technology, we cloned11PmsHSPs genes from ’Xue Mei’. Through Blast comparison and cluster analysis of amino acids, The structures of different PmsHSPs were very similar. The sites of PmsHSPs’ intron insertion were in two special particular sites. Few cytoplasm PmsHSPs had introns while most organelle PmsHSPs had introns. This experiment Preliminary discussed the system evolution, the function of introns and the formation, function and control means of PmsHSPs.3. The primary function analysis of sHSP. The sense expression vector of PmHSP18.0-C Ⅰ, PmHSP17.9-CⅡ, PmHSP17.6-CⅢ was constructed by ligating the cloned fragment to sHSP genes and were transformed into abrabidopsis through inflorescence After three generations of cultivating, we got the seeds of high homozygous rates. The growth condition of these seed were then observed under different heat treatment. The result showed that growth condition of the transgenic plant were much better than the wild plants and there roots were stronger.