Cloning And Function Analysis Of Thermo-sensitive Male Sterile Gene TMS10 In Rice(Oryza Sativa L.)

The thermo-sensitive male sterile rice plays a crucial part in two-line hybrid rice breeding and is an important material for a study on male gametophyte development.Male gametophyte development undergoes anther differentiation,tapetum apoptosis,pollen wall formation and fertilization of continuous development,which is a key on the basic theory in hybrid rice breeding and reproductive development research.Any gene mutations involved in these processes can cause anther dysplasia,eventually leading to male sterility.Pollen wall construction is a very complex and orderly process.It contains the development of callose walls,intine,nexine and so on.They influence each other,involving the sporophyte and gametophyte interaction.The process is strictly controlled by multiple genes.The molecular mechanisms underlying the formation of the pollen wall remains largely unknown,and it still has a lot of doubts to solve.In this study,we characterized the a new rice temperature sensitive male sterile mutant,named TMS10,which showed a defective pollen wall and complete male sterility.TMS10 encodes a acyltransferase(Omega-hydroxypalmitate O-feruloyl transferase /Omega-hydroxyacid O-hydroxycinnamoyl transferase,HHT).By methods such as function complementary experiments,we try to confirm TMS10 encoding HHT and reveal the molecular regulation mechanism of TMS10.The main contents and the results are as follows.1.At the same Illumination time of 13 h,undered the control of high temperature(an average temperature of above 28?),tms10 is completely sterile.However,with the low temperature conditions(the average temperature is 22?),tms10 is fertile.It suggests that the pollen fertility of tms10 is regulated by temperature.2.Anther development of tms10 has been observed through semi-thin and ultra-thin section technique.Comparing the cytological characteristics between tms10 and PA64 during development,our research found some abnormalities of tms10,when it is treatmented by high temperature.Main abnormalities are: the less deposition of callose walls,the thinner of tapetum,incontinuous but thicker nexine and defective pollen.With low temperature treatment,tms10 restores pollen viability and fertility,and the basic structure of pollen is the same as PA64.3.Based on the candidate gene sequencing,there was a point mutation among Zhonghua11,AN,tms10,from G(guanine)to T(thymine),leading to amino acid from arginine(Arg)mutations for Leucine(Leu).Bioinformatics analysis found that the protein contains two conserved domains HXXXD and DFGWG,which belongs to the contained the conserved BAHD acyltransferase family.Moreover,its function is similar to the Arabidopsis' s HHT.4.The functional complementation vector,overexpression vector,rnai vector,Cas9 vector of TMS10 were constructed and the transgenic plants were obtained by agrobacterium-mediated method.The functional complementation vector were transformed tms10 and the latter two were transformed Zhonghua11.The functional complementary transgenic plants(tms10-FC)and overexpression transgenic plants(tms10-OE)fertility is restored.The pollen fertility of rnai transgenic plants(tms10-rnai)decline and the pollen of cas9 transgenic plants(tms10-CAS9)abort and cannot be completely restored after low temperature treatment.These TMS10 encoding HHT regulation of thermo-sensitive male sterile rice.overexpression plant.It revealed that thermo-sensitive male sterile of tms10 is regulated of TMS10,encoding HHT.5.The anther development of tms10-CAS9 and Pt(TMS10 transformed PA64)plants has been observed through semi-thin and ultra-thin section technique.Without middle-level degradation,tapetum degradation of tms10-CAS9 is delayed,eventually forming the shrinkage defects of pollen grains.At stage S10 the middle layers of Pt is swollen and cuticle structure is obvious.The middle layers of Pt degrade at last to form defective mature pollen grains.Among Pt,Zhonghua11,tms10-CAS9 transgenic plant,cuticle structure appear on anther's epidermis first but thinner at stage S13 in Pt.With defective pollen as the same as tms10,tms10-CAS9 transgenic plant and Pt plant appear other abortive features,which suggest that pollen fertility also affected by other genes.6.The result of qRT-PCR showed that TMS10 expressed in root,sheath,and leaves of the two-week rice seedling and anther,which expressed lowest in young leaves.at stage S13 the expression of TMS10 in anther with high temperature treatment is 2 times higher than that in temperature low temperature.Expression of TMS10 in anther is rise from stage S5 to S8 and then decrease,with the highest expression at stage S8 and with the lowest expression at stage S12.7.In order to explore subcellular localization of TMS10,we constructed transient expression vector which fused the full ORF sequences TMS10 gene to the GFP gene and transformed the transient expression vector into the protoplasts and bombardment-mediated transient expression into leaf sheath,the results showed that the TMS10 targeted to the cytoplasm.8.In order to detect the changes of chemical composition in the pollen wall,a lipophilic dye Sudan red 7B and Fluorol Yellow 088 were used to staining the mature pollen grains respectively,which showed that lipid content decreased but suberin composition improved.Observing Phenolic compounds of mature pollen grains in the ultraviolet,we found phenolic compounds increased.In summary,this study proves Thermo-sensitive male sterile gene TMS10 in rice is required for the anther polyester synthesis,which involves in the formation of pollen extine.Distruption in this process might result in abnormal microspore development process,and ultimately lead to pollen abortion.These results have important significance in illustration of anther development and anther related lipid metabolism in rice,and provide a new genetic resource for molecular breeding based on the utilization of thermo-sensitive male sterile in rice.