Expression And Function Analysis Of Candidate Genes For Multi-glume Mutants In Rice (oryza Sativa L.)

The development of floral organs is closely related to the yield and quality in rice,but its genetic mechanism is still unclear.Our research group found two mutants with completely degenerated stamens and carpels(named as pistil-stamen degeneration 1 and 2, psdl and psd2) from rice breeding materials.By fine mapping,a MADS transcription factor gene was identified to be the candidate of PSD1 and two genes encoding triacylglycerol lipases(named as triacylglycerol lipase 1 and 2,TG1 and TG2) were identified to be the candidate of PSD2.In this study,on the basis of above results,the complementary vectors,over-expression vectors and RNA interference vectors of the two candidate genes of PSD2 were constructed and were used to transform rice.Some transgenic plants were acquired.In addition,the developmental processes of floral organs of psd1 and psd2 were observed through scanning electronic microscope(SEM) and the temporal and spatial features of expressions of PSD1 and PSD2 were analyzed using in situ hybridization.This study will facilitate elucidating the functions of PSD1 and PSD2.The major results are as follows:1.Two complementary binary vectors of the two candidate genes of PSD2 were constructed,named as pCV-TG1 and pCV-TG2,pCV-TG1 was constructed by ligating the promoter(1954bp) and open reading frame(ORF) of TG1 with modified pCAMBIA1301. pCV-TG2 was constructed by ligating the complete TG2 gene(including the 803bp promoter) sequence with pCAMBIA1300.Both pCV-TG1 and pCV-TG2 were used to transform mutant psd2 and some transgenic plants were obtained.2.Two over-expression vectors of the two candidate genes of PSD2 were constructed, named as pOV-TG1 and pOV-TG2.They were constructed by inserting the ORFs of TG1 and TG2 into the multiple cloning sites downstream of the 35S promoter in modified pCAMBIA1301,respectively.Both pCV-TG1 and pCV-TG2 were used to transform the wild type rice and some transgenic plants were obtained.3.Two RNA interference vectors of the two candidate genes of PSD2 were constructed,named as pTCK-TG1 and pTCK-TG2.The lengths of the two candidate genes separately were 580bp and 255bp.Both pTCK-TG1 and pTCK-TG2 were used to transform the wild type rice.25 transgenic plants of TG1 and 24 transgenic plants of TG2 were obtained.And the latter ones of which 5 were abnormal.One side of the degenerated glume is longer than the wild type.It is similar to eg mutant and different from the pheonotype of psd2.So we elementarily postulate that TG2 is not the gene of PSD2.4.The results of scanning electronic microscope(SEM) suggested that the staman anlagen and carple primordia were completely degenerated and replaced by lemma and palea promordia.Their differences are that aditional glumes promordia of psd1 mutant is rotated with six anlagen when the psd2 mutant is symmetrical with two ones.5.The results of in situ hybridization suggested that expression of PSD1 was first detected at the time when lemma and palea priomdia were just initiating.At this stage, strong expression of PSD1 was observed mainly in its central anlage.From the lodicule anlage,the stamen anlage and the capel anlage startting to split up to completing the differentiation,PSD1 continues to express in the stamen,but expresses by weakly to stiffen in the lodicules and the capel.After the floral organs have formed basically,it still could examine the strong expression in the lodicules,stamens and capels.But expression of PSD2 was initially detected at the time when lemma and palea had differentiated and the lodicules and stamen priomdia was initiating.PSD2 has the expression in the entire floret anlage,weak expression was detected in lemma and palea priomdia but in the lodicule anlage and the undifferentiated third,fourth whorl has the strong expression quantity. PSD2 was persistly expressed in the primordia of stamens and carpels from their inception. After the floral organs have formed,no siganal was detected.