Analysis On Progeny (T4) Of Lysine-rich Protein Gene (sb401) Transgenic Rice And Genetic Mapping On An Excessive Tillering Gene In Rice

Rice is one of major food of our country.Along with in-depth research at resistance and output,we are paying more and more attention to the increase on quality of rice,one of the index of which is the content of protein and amino acids.But the average content of protein is only 6.3%～7.1%in edible milled rice,which is the lowest in cereal crops.The lysine content is only 0.22%～0.28%,which is considered to the first limiting amino acid.Using transgenic technique to modify rice quality has become an important method today. How to get the material with constant foreign gene expression and heritability has become a research focus.Thermal asymmetric interlace PCR was invented by liu,et al.It become an important method of reverse genetics quickly because of its extensive advantages.At the same time,it also become an important method on foreign gene research.Our research material is Nipponbare(Oryza sativa ssp.japonica) into which sb401 gene was successfully introduced by Agrobacterium tumefaciens-mediated transformation.We systematially analyzed integration site and expression rule of foreign gene of different transgenic lines with southern blotting,TAIL-PCR,and seed total protein and amino acid assay.The research results are as follows:1.nine independent homozygous T4 generation sb401 transgenic lines were obtained through consecutive selfing and detecting.2.Through the southern blotting analysis on the transgenic lines,we found that all the transgenic lines show 1～3 bands except the negative control and the integration sites were different from each other.The results showed that the foreign gene were integrated into the genome with single or low copy through Agrobacterium tumefaciens-mediated transformation.3.After TAIL-PCR analysis of the T-DNA of the nine transgenic lines and sequencing of the cloned fragments,we got eight flanking fragments(containing Nipponbare(Oryza sativa ssp.japonica) gemone sequence),which were located on seven chromosomes of japonica rice.The result was just the same as that of southern blotting that the integration sites of foreign gene were different.The results of flanking sequence of the two lines were obtained through TAIL-PCR walking,which also confirmed the existence of atypical integration models of T-DNA.4.The result of a detection of protein and amion acid of nine homozygous transgenic lines (with the non-transgenic line as the control),showed that the total protein increased by 2.7%～18.4%,amino acid by 2.3%～15.8%,lysine by 0%～10%.all of those results indicated the expression of sb401 gene in all the transgenic lines(except LK3).However,the analysis also showed that the increase of total protein content,amino acid content of transgenic lines was not positively proportional to the lysine content.It showed that sb401 gene played an increasing role in different amino acid components.5.Analysis combining blotting,TAIL-PCR and protein detection showed that the expression difference was not obvious when it comes to low copies.Besides,the relationship between the integration sites and copy number was discussed.Rice is one of the major food sources,which is third to wheat and maize in the world total production of food grains.Tillering in rice(Oryza sativa L.) is an important agronomic trait that affects grain production,and it is pointed out that tillering number should be moderate in super-rice.As a special kind of branch of monocotyledonous plants,rice tillering is also of developmental importance.Therefore,tillering of rice is one of the focuses in rice genetic and developmental research.A rice mutant with an excessive tiller number,designated ext-M 1 B,was found in the F₂ progenies generated from the cross of M1B and GMS-1(a genetic male sterile),whose number of tillers was up to 121.The crosses from M1B-ext/M1B,M1B/M1B-ext,2480B/ M1B-ext,D62B/M1B-ext,G46B / M1B-ext and G683B / M1B-ext expressed normal tillering in F₁,and segregated into the two different phenotypes,normal tillering type and excessive tillering type in a ratio of 3:1 in F₂.At the same time,it was found that the phenotypes of excessive tillering and normal tillering plants in F₂ populations were similar to that of M1B-ext and another corresponding parents with normal tillering respectively,and there was no tall individual in the excessive tillering plants and no dwarf individual in the normal tillering plants.These results indicated that the excessive tillering trait of ext-MIB is controlled by one recessive nuclear gene.The F₂ population of 2480B / M1B-ext was used as the mapping population of the mutant gene.It consisted of 602 recessive excessive tillering individual plants and 1865 homologous dominant normal tiller number individual plants.The linkage analysis results found four linkage markers,LKW3,LKW34,LKW97 and LKW84,After constructing the regional molecular linkage map of the ext-M1B gene with the MAPMAKER software,the four makers were located on the short arm of rice chromosome 6,and the genetic distance from the target gene to the markers,LKW3,LKW34,LKW97 and LKW84 was 5.0cM,1.2 cM,3.8 cM and 5.1 cM,respectively.