EMS Induced Pyrus Betulaefolia Dwarf Mutants And Research On Dwarf Related Genes

In order to obtain Pyrus mutants with better traits and to study the genetic mechanism of dwarf trait of pear,the ethyl methane sulphonate(EMS)mutation breeding system for Pyrus betulaefolia was built.GA20 gene from pear cultivar ’Aizhen 51’and the dwarf gene GAI from Arabidopsis were cloned.Then we researched gene expression and genetic transformation.The results are as follows:1.Seeds of Pyrus betulaefolia after stratification were treated with different concentrations of EMS to induce mutation.Then the germination percentage of seeds,plant height and intermode length of seedlings were investigated.The genetic variation sites were identified using SRAP markers.Then it was confirmed that the concentration of EMS treatment for half lethal dose of Pyrus seeds is 0.3%.The optimum treatment for increasing mutation is 0.4%of EMS,and the mutation rate is 37.3%.Phenotypic data revealed that plant height with different EMS treatment were significantly lower than the CK group,and the intermode length was 4.1%～17.4%shorter.These results provide basic theory for obtaining dwarf pear mutant effectively.2.DNA from young leaves of pear cultivar ’Aizhen 51’ was extracted and gDNA sequence of GA20ox was cloned using homologous amplification method.Sequence alignment analysis revealed that gDNA sequence of GA20ox from ’Aizhen 51’ was 1899 bp long with two introns and three exons.Sequence identity of GA20ox from ’Aizhen 51’and’Dangshansuli’ was 98.16%,and few nucleotide substitutions were identified.Meanwhile,the promoter sequences of GA20ox from ’Aizhen 51’and ’Dangshansuli’ were also cloned and the cis-acting elements of them were analyzed using PLACE software.The results revealed that both promoters comprised two core promoter elements(TATA-BOX)in the positions of 1144 bp and 1429 bp,respectively,and also contain some other cis-acting elements regulating and controlling gene expression.We also identified few nucleotide substitutions between the two promoters.RNA from young leaves of ’Aizhen 51’ pear was extracted and it was reverse transcripted to single stranded cDNA.Overall sequence of dwarf related gene GA20ox was amplified using RT-PCR.Length of GA20ox was 1164bp,encoding 387 amino acid.Sequence alignment analysis of GA20ox from ’Aizhen 51’ with genes from other species revealed that GA20ox from ’Aizhen 51’ and gene from ’Zhongai 1’(Acc.HQ833589)were with closest genetic relationship than others.GA20ox from ’Aizhen 51’have a distant genetic relationship with gene from Gossypium hirsutum(Acc.ACM68924.1).RNA from pear cultivars ’Aizhen 51’,’Dangshansuli’,and ’Zhongai 1’ from different growth and development periods was extracted,and expression analysis of them was performed using real-time fluorescent quantitative PCR.Result analysis revealed that lower expression of GA20ox gene in the middle of vigorous growth period caused dwarf trait of’Aizhen 51’.3.For innovating the dwarf plants of pear.RNA from young leaves of Arabidopsis was extracted,and it was then reverse transcripted to single stranded cDNA,overall sequence of dwarf related gene GAI from Arabidopsis was amplified using RT-PCR.Sequence identity revealed that amino acid sequence of this gene was totally accordance to the Arabidopsis GAI gene that retrieved from genbank,which proved it was the target gene.The over expression vector was constructed by transferring GAI gene into pBI121 vector.According to the published genetic transformation system of pear,we optimized the genetic transformation system of Pyrus bretschneideri and transfered GA1-pBI121 vector to Pyrus bretschneideri using freeze-thaw method.But we didn’t get the transgenic plants yet.