Site-directed Mutagenesis Of Genes Associated With Grain Weight In Rice

Rice is one of the important food crops in China and all over the world.As the growth of the population,the decrease of cultivated land area and the industrialization of the change of natural environment,food supplies become a pressing problem.Therefore,raise the level of rice yield is one of the important goals of rice breeding.As the development of rice functional genomics research and genome editing techniques,Make it possible for important agronomic traits of rice gene on directional improvement in the molecular level.This study aimed to use the latest genome editing techniques——TALENs and CRISPR/Cas9,the two negative regulation of rice grain weight of zinc finger protein gene point mutation,And through the mutant offspring selfing screening out big grain of rice new material of exogenous DNA sequence.The main research results are as follows:1?Analysis of 28 varieties of GW2 gene cDNA sequence.Results indicate that,In the detection of rice varieties,The GW2 gene exon 1 and exon 8 respectively exists a SNP loci.Analysis found that the size and the shape of them with the rice grain weight had no obvious correlation.But,Big grain varieties 1g-3 of Grain length and width is significantly large,The first beetle found the GW2 gene show 357th a base mutation,Lead to early termination protein translation.So consistent and predecessors' studying results,Loss of function the GW2 gene can increase grain weight.2?Based on the above sequence analysis results,We at first GW2 gene exon respectively constructed two TALENs expression vector,Ming hui 86 and xiushui 134 and Nipponbare for transformation receptor material.Through the agrobacterium mediated rice genetic variations,obtained respectively 11 and 26,27 positive transgenic cloning regeneration plant;Target gene by PCR amplification and sequencing method,Only in Minghui 86 get three strains mutant which GW2 gene in first exon missing.The Numbers for G3 for missing 90 bp homozygous mutant,The Numbers for G14 and The Numbers for G19 are hybrid mutants respectively missing 9 bp and 32 bp.3?Extract the G3 and G19 mutant plant leaf RNA,RT-PCR amplification GW2 gene cDNA and sequencing analysis,GW2 gene of G3 in the mutant to frameshift mutations,Cause changes in the protein structure,G19 mutants in GW2 gene produce early termination codon,Make loss of protein function.4?Comparison of wild type analysis of mutant G3 and G19 of TO generation since the solid grains,Found the G3 homozygous mutant in grain length,width,and grain weight increased significantly,G19 hybrid mutants than controlled grain length and grain weight increase,But there is no obvious change grain width,Further analysis T1 generation,G3 grain traits are not isolated,G19 grain traits to obtain three kinds of separation,Showed the GW2 gene to the influence of the grain kernel weight may exist dose effect.In addition,the T1 generation,By PCR detection were obtained without exogenous DNA sequence G3 homozygous mutant 23 strains,G19 homozygous mutant 2 strains,G19 heterozygous mutant 4 strains.5?Construct three TALENs vector for OsTZF9 gene,Agrobacterium large-scale transformation of rice callus,Material to restore 86 and xiushui 134,By hygromycin selection,PCR,sequencing selection for transgenic plant 87 strains were positive,But not detected by gumming point missing plants.Currently building 3 CRISPR/Cas9 expression vector,The transformation has been get resistant callus.