Cloning And Preliminary Functional Analysis Of Self-incompatibility Gene In Plant

Object: Self-incompatibility (SI) is the prevalence of phenomenon in the process of plantreproduction,it is an intraspecific reproductive barrier adopted by angiosperms that allows thepistil to distinguish between self (genetically related) and non-self (genetically unrelated)pollen.The identification of self-incompatibility is an extremely complex process, involved inthe recognition of S-RNase and SLF in GSI. It was postulated that an SLF allelic productspecifically detoxifies its non-self S-ribonucleases(S-RNases), there are also other cofactorsto participate in the coordination of this complex process.Self-incompatibility in floweringplants keeps the good varieties of crops and improves the production of seeds. In this study,we cloned the related genes, For providing an important genetic resource in breeding.Methods:(1) The clone of related genes in SI: Wild-type self-incompatible lines were solanaceaePetunia inflata. For its great variety, the genotype of materials are uncertain.According toknown petunia’s S-RNase and SLF in Genebank, A gene of S-RNase and SLF was clonedfrom Petunia hybrida cDNA By using some internet datebases and biological softwares suchas NCBI, CODEHOPE, Primer Premier5.0to design degenerate primers and degenerateprimers RT-PCR.(2) the clone of LAT52: Extraction of tomato genomic DNA, according to the knownsequence of LAT52, design specific primers, the pollen specific promoter LAT52was cloned.(3) The clone of Barnase and Barstar in Bacillus amyloliquefaciens: Extraction ofBacillus amyloliquefaciens DNA, according to the known sequence of Barnase and Barstar,design specific primers, the gene of Barnase and Barstar was cloned.At last, we constructed the vector,and transformed pBI121-LAT52-sx into Arabidopsisand Tobacco,identificated transgenic plants with PCR, compared with wild type andtransgenic plants.According to the pollen’s TTC colouration,we preliminary analyzed thefunction of sx gene. At the same time, transformed pBI121-Ubi-epsps-LAT52-sx intoeconomic crops Mentha haplocalyx, preliminary analyzed the sterility.Results:(1) A417bp fragment was cloned from Petunia hybrida cDNA By using some internetdatebases and biological softwares such as NCBI, CODEHOPE, Primer Premier5.0to designdegenerate primers and degenerate primers RT-PCR. Through analysis in GenBank, It has thehighest similarity with SX gene sequence. Designing a specific primer according to thesequence of SX gene, finally a747bp fragment was obtained by RT-PCR. The result showed that SX was660bp in size and encoded220amino acids, including a transmembrance signaldomain of22amino acids, the conserved regions(C1-C5)and hyper-variable regions(HVa andHVb).(2) According to the gene of SLF, comparing homologous gene conservative region withCLASTALX, using specific primers, a1200bp fragment was cloned, which named SLF. Itwas found that It has the highest similarity with Sn-SLF3gene with DNAMAN, and aminoacid sequence was similarity with S7-SLF3, was86.63%. According to Kubo etc. analysis ofpetunias pollen SLF amino acid homology, found it has the same F-box area.(3) An anther-specific gene of717bp from tomato was isolated and expressed. Comparedwith the GenBank LAT52, nucleotide similarity is99.44%,7redundant bases and2mutativebases in non-critical area.7redundant bases AAAAAAT is a simple sequence repeat, there are4repeat sequences in this mutations, added a registration sequence than GenBank.(4) two gene of348bp and367bp was cloned from Bacillus amyloliquefaciens,constructpBI121-LAT52-BN, and expected to converse into Arabidopsis thaliana compared withpBI121-LAT52-sx.And then a signal peptide of22amino acids was removed to make it has a function ofS-RNase in pollen, the pollen-specific LAT52promoter of tomato was used to express sx,anda plant expression vector was constructed. Transgenic Arabidopsis plants expressing sx caninfluence the development of Arabidopsis’s floral organ, the pollen vitality is very low andeven no vitality, and cause the infertility of transgenic as a result.At the same time,transformed pBI121-Ubi-epsps-LAT52-sx into economic crops Mentha haplocalyx,TTCcolouration shows that the pollen vitality of transgenic herbicide is very low and even novitality, and cause the infertility of transgenic as a result.In this experiment,we transformedherbicide-resistant genes and sterility in integrated into Mentha haplocalyx.For Menthahaplocalyx and even of all the economically important plants on the varieties of fine breedingprovides an important genetic resources.