Cloning And Preliminary Functional Analysis Of A Seed Size Gene In Maize

To increase the yields and quality of food crops is an important aim in plant science research.Corn is an important food crop and forage crop,in many graminaceous crops such as corn,seed size is a very important yield trait.Maize yield is decided by three factors of ears per acre,grains per spike and grains weight.Among these,grains weight is determined by seed size and grain test weight,the grain size plays a decisive role in grains weight.Seed can be split into two main biological components,embryo and endosperm,endosperm accounted for about 70% of the total weight of grain seed and makes a great influence on seed size and grains weight.So the most important factor to analysis the mechanism of the seed growth and development is discovering the key mutation gene that controls the seed growth and development.This study used a natural mutant 6244 as basic material,which is an endosperm cell number and seed size mutant and found in the process of breeding,we built a segregation population with B73 and exploited many functional markers,succeed with the fine mapping of target gene,we also determined the candidate gene through sequencing analysis of mutant and normal individual.The rusults we have got were as follows:1.Through cross and backcross with inbred line B73 and genetic analysis of segregation posterities,we found the mutation phenotype is controlled by a gene's recessive mutation and belongs to the classic Mendelian inheritance,then we mapped this gene on 1.02 bin of maize chromosome 1.2.According to previous research results,Zm Es M1 gene was mapped on chromosome 1 between bnlg1614 and bnlg1953 on 1.02 bin by SSR based on B73 sequence.To fine map Zm Es M1 gene,a larger segregation population with 40,000 plants was performed.500 primers including SSR marker,insertion/deletion marker,etc,were exploited to increase the molecular marker density in 1.02 bin and 45 primers were identified as polymorphism.Finally,the Zm Es M1 gene was fine mapped between marker N-85 and N-15,the physical distance is about 120 kb.3.Analysis the base sequence between N-85 and N-15 by FGENESH,we have predicted three coding genes in this region,the No.2 gene is the preferential candidate gene and it's predicted function is about cell division cycle.Then we did a sequencing analysis of No.2 candidate gene between B73 and 6244,it turned out that the mutational parent had two more insert sites than normal parent in the promoter region,the insert fragment sequence is GAATCATG and TTATG.There are also two SNPs in coding region,the mutation from G to C in the second exon and from T to C in the fifth exon,and their corresponding encoded amino acids have also changed.Then we planned to construct the expression vector of No.2 candidate gene for genetic transformation.