Fine Mapping Of Male-sterile Gene Ms39 And Genetic Transformation The Development Related Genes In Maize

Maize is a staple crop used for food,fodder and biofuels,it is important to increase the yield and biomass in maize research.Because maize shows strong heterosis and male sterility is a common phenotype which was valuable for hybrid production in maize,male sterility has earned widespread attention by breeders.In addition,vegetative growth is the prerequisite for reproductive growth in plant.The plant vegetative growth can be divided into juvenile stage and adult stage,the process of transition from juvenile to adult vegetative growth is defined as vegetative phase change.The vegetative phase change is a complex process that regulated by multiple genes which are important for reproductive phase change that has a significant guiding in maize produce.This research work is composed of fine mapping the male sterility gene ms39 and genetic transformation of the development related genes in maize.A maize male sterility mutant was obtained and identified from outer space flight in previous study,named ms39.In this research,our main work is fine mapping male sterile gene ms39 and related research.First of all,we characterized the phenotype and performed the cytological observation of anther in mutant.Then 1,073 male-sterile plants from（ms39×Mo17）F₂ were used as mapping population,ms39 was finally mapped by continually finding and developing new molecular markers.After that,then genic molecular markers were deigned to detect the results of fine mapping.The main conclusions were listed below:（1）Analysis of plant height,ear height,tassel branch number,ear length and stem diameter in different segregated populations showed that the male-sterile plants were significantly lower than male-fertile plants.（2）Cytological observation were performed by paraffin section for microsporogenesis of ms39 mutant in advanced sibling population.The results illustrated that the abnormal anther development started from dyad stage of meiosis in ms39 mutant,which showed autolyzed phenomenon of dyad and disordered arrangement of tapetum cells,finally,tetrads cannot form microspores.（3）According to increase marker density and narrow mapping interval using 1,073male-sterile plants from（ms39×Mo17）F₂,ms39 was finally mapped in an interval between marker L7 and M30 with physical distance 365 kb.Two genic molecular markers,named ARF12-3-2 and NACTF82,were developed in 365 kb interval and finally the ms39 was mapped in L7 and NACTF82 with physical distance 319kb.（4）Between L7 and NACTF82,ten candidate genes were predicted.After cloning and sequencing the CDS of ten genes,it was showed that a 4-bp deletion in coding region of gene Zm00001d043909（Cals12）in male sterile mutant.（5）A genic specific marker InDel-cals was designed according to a 4-bp deletion of Zm00001d043909（Cals12）.Using InDel-cals to detect the male-sterile plants from four segerated population,117 male-sterile plants from_ms39×B73BC2F2 population,320male-sterile plants from（ms39×Mo17）BC4F2 population,340 male-sterile plants from（ms39×Mo17）BC5F2 population and 170 male-sterile plants from sister cross population,no crossover was detected which indicated it was complete linkage between gene Zm00001d043909Cals12</sub>and ms39.（6）Compared with normal plant,there was a 4-bp deletion in coding region of candidate gene Zm00001d043909（Cals12）in ms39 mutant.The results of predicting the protein with DNAman software showed that this 4-bp deletion can cause early termination of protein translation and resulted in non-functional Cals12 protein.In addition,according to the bioinformatic analysis found two genes which were predicted to regulate vegetative phase change,ZmNF-YA-10（Nuclear factor,have CCAAT binding site）and ZmNovel-TF（lack of annotation）.In order to understand the founction of two genes,pANIC8D-ZmNF-YA-10-RNAi and pANIC8D-ZmNovel-TF-RNAi interference vectors were respectively constructed.By Agrobacterium tumefaciens mediated transformation,vectors were transformed to maize line Hi-II to obtain positive seedlings.Then we compared the phenotypic difference of last leaf with epicuticular wax（LLEW）and total leaf number.The main conclusions were listed below:（1）From statistical analysis,the average of LLEW and total leaf number of transgenic plants were significantly higher than that of non-transgenic plants（7.63±0.49 VS7.29±0.25,P&lt;0.05）,（15.28±1.41 VS 14.67±0.21,P&lt;0.05）.The average LLEW of T2-generation transgenic plants cultivated in greenhouse were significantly higher than that of non-transgenic plants（6.91±0.0015 VS 6.61±0.007,P&lt;0.05）.The above results indicated transforming ZmNF-YA-10-RNAi can extend transition time from juvenile to adult of maize vegetative growth.（2）We also compared the average of LLEW and total leaf number between ZmNovel-TF-RNAi transgenic plants and non-transgenic plants of T1 generation.The results showed there no significant difference of LLEW and total leaf number between transgenic and non-transgenic plants.There is also no significant difference between transgenic plants and non-transgenic plants of T2 generation cultivated in greenhouse.In summary,ms39 gene was mapped in a region of 319kb between markers of L7 and NACTF82.After sequencing all candidate genes,we predict Cals12 is the key gene to regulate ms39 mutant,which encoded the callose synthase 12.This results lay foundation for cloning the ms39 gene.In the process of anther development,callose surrounds the pollen mother cells and tetrad for supporting and protecting microspore.Therefore,our study not only provides resource for the research of maize male sterility but also enriches regulation pathway of maize male sterility.Using the RNAi technology by transforming interference vector pANIC8D-ZmNF-YA-10-RNAi and pANIC8D-ZmNovel-TF-RNAi,we found the average LLEW of offsprings of transgenic plants was higher compared to that of non-transgenic plants which indicated reducing expression of ZmNF-YA-10 can extend transition time from juvenile to adult stage of maize vegetative growth.After transforming interference vector pANIC8D-ZmNovel-TF-RNAi,we did not observe corresponding phenotype in offsprings.</sub>