| Chalcone synthase is a key enzyme of flavonoid biosynthesis pathway.In the study,we choosed Betula pendula ’Purple Rain’ as material,cloned a chalcone synthase gene(BpCH3)and analyzed expression features of the gene family.BpCHS3 overexpression transgenic birch were obtained by using the Agrobacterium-mediated system,and then we analyzed drought resistance and salt resistance of transgenic lines.The results of the study were as follows:(1)The total length of BhCHS3 gene was 1170 bp,encoding 389 amino acids,protein conformation stability,containing seven disulfide bond,the relative molecular weight is 42556.2 D,did not contain function structure domain;The formulas for the gene was C1896H3037N505O563S20,theory of isoelectric point was 6.18,the residual positive and negative charge were 47,43,respectively.The evolutionary tree that contained BpCHS3 amino acid sequence and other CHS amino acid sequence of 14 species showed the homology was the highest in BpCHS3 and Casuarina CgCHS amino acid sequence.(2)The leaf color of purple rain birch changed from purple to green in time and space,as the leaf colour from purple to green,anthocyanins content gradually decreased,content of chlorophyll gradually rose.Real-time PCR was used to analyze the spatial and temporal expression pattern of BpCHS and BpDFR family genes.The results suggested that,the temporal expression regularity of two gene family was obvious in upper leaves of purple rain birch,expression gradually reduced from May to July,August and September,expressing quantity began to gradually pick up.In August,the color of leaves presented obvious stratification in purple rain birch,BpCHS,BpDFR gene family express higher in the upper leaves and express lower in the middle and lower leaves.The expression of two gene families in the purple rain birch was higher than European white birch(CK),the expression of BpCHS3 gene was the largest quantity than CK.(3)The engineering bacteria EHA105(pCHS3)was acquired by gateway method,then 15 BpCHS3 transgenic resistance strain were acquired by agrobacterium mediated zygote embryo catalysis to genetic transformation of birch,under 50 mg/L of hygromycin selection.PCR and qRT-PCR suggested that target gene had been successfully transferred into birch and could be expressed stablely.The expression of BpCHS and BpDFR gene family in transgenic birth was higher then that in NT.(4)Drought and salt resistance analysis suggested that drought resistance and salt resistance of transgenic lines significantly enhanced.Under 0.30%NaCl stress,the rooting rate of transgenic lines was 100%after 25 days,the average number of each taproot is 2～3 times of NT,the average taproot length was 6～7 times of NT.Under 0.50%NaCl stress,the rooting rate of NT was 0,salt injury index was 1.0,while the rooting rate of ransgenic lines was still 100%,salt injury index was about 0.43～0.48.Under 5.0%PEG treatment,the rooting rate of NT was only 30%after 30 days,drought injury index was 0.93.The rooting rate of transgenic lines was between 80%～90%,which was about three times of NT,and the average number of each taproot was about three times of NT,drought injury index was low,respectively 0.18 and 0.43;When the concentration of PEG was 7.5%,NT were all dead,while the rooting rate of transgenic lines was 60%～80%,most of the plants growed well. |
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