Study On Endosperm Development And Related Gene Expression After Interploidy Crosses Between 2N And 4N Rice

Many flowering plants are polyploid, but crosses between individuals of different ploidies produce seeds in particular the endosperm that develop abnormally. On the study of Arabidopsis, it was found that the differences of endosperm produced by interploidy crosses were relevant with the genomic imprinting. Genomic imprinting is a phenomenon that the gene is expressed primarily or exclusively from either the maternal or paternal allele and plant imprinting has been described primarily in endosperm. It plays a role in regulating endosperm development and affecting seed size. Rice(Oryza sativa L.) is one of the most important crops in the world and its endosperm is the main part of human consumption. The endosperm development and expression pattern of imprinted genes are different between rice and Arabidopsis. But the genomic imprinting involved in regulating endosperm development and the seed size has not been paid enough attention. The Nipponbare (2n), Haitian(2n) and DHaitian(4n) which have SNPs differences were used in this experiment to obtain endosperm with different ploidies by interploidy crosses between subspecies of rice. The endosperm development was observed. The relationship between the parental genome dosage and imprinted gene expression was studied by RT-PCR, SNP, Sub-clone and sequencing. In addition, because genomic imprinting belongs to the category of epigenetics, some genes which were involved in epigenetic modification were quantitatively analyzed. These analyses were trying to uncover the relationships between genomic imprinting, imprinted gene expression and seed development.The main results were as follows:1. The observation of the endosperm development after interploidy crosses:(1) The results of pollen stainability identified with the method of I2-KI revealed that Nipponbare (2n) pollen stainability was the highest(63.2%), Haitian(2n) took the second place(61.54%),while the D Haitian(4n) was the lowest(33.91%).(2)The germination and elongation of pollens were observed with ovaries cleared by KOH and stained by aniline blue after interploidy crosses and the results showed:all the pollens after pollination could germinate and went deep into the bottom of the ovules.(3)The obversation of endosperm development after interploidy crosses with two methods of whole clearing-DIC and paraffin sections revealed that:the endosperm development was different from each cross. And the differences became most obvious on the third day after pollination:the endosperm had already finished cellularisation in the controlled combinations(2n X 2n and 4n X 4n), while the endosperm produced by 2n×4n was still in the syncytial period without cell wall aroud the free nuclears. Although the endosperm produced by 4n×2n has also finished cellularisation, the cells'number was smaller and they were closely together.2. The research for the relationship between the rice parental genome dosage and the imprinted gene expression by means of RT-PCR, sub-clone, sequencing showed that the relationship was very complex and it could be divided into three types:①Imprint completely:the imprinted genes were free of parental genome dosage regulation, such as paternal imprinted gene Os07gl7460;②Imprint not completedly: when the paternal or maternal genome dosage increased and the maternal or paternal genome dosage kept unchanged, these imprinted genes lost imprinting, such as maternal imprinted gene Osllg27470;③Completedly not imprint:the imprinted gene could lose imprinting when the genome dosage from either the male parent or the female parent increased, such as paternal imprnted gene Os08g27240.3. Though testing the expression proportion of the imprinting, the results showed that:①Imprint completedly:for example, subclones of the paternal imprinted gene Os07g17460's RT-PCR products conformed that all the hybrid endosperms only contained mRNAs from the male parent;②Imprint not completedly:taking the paternal imprinted gene Os10g04890 for example, there only contained mRNAs from the male parent in the endosperm produced by the control combination and D Haitian(4n) X Nipponbare (2n), while there were mRNAs from both the parents in the endosperm produced by Nipponbare(2n) X D Haitian(4n) and the proportion was 16:13(close to 2:2);③Completedly not imprint:taking the paternal imprinted gene Os08g27470 as an example, there only contained mRNAs from the male parent in the endosperm produced by the control combination, while there were mRNAs from both the parents in the endosperm produced by Nipponbare(2n)×DHaitian(4n) and D Haitian(4n)×Nipponbare (2n).the proportions were 23:2 and 19:8 respectively.4. The analysis on quantitative expression of rice PcG genes(such as OsFlE1,OsFIE and OsEMF2) and DNA transmethylase gene OsMETlb which participated in epigenetic modification using QRT-PCR discovered that:compared with the controlled combination(2n X 2n), the expression level of OsFIE1 and OsFIE2 increased strikingly while OsMET1b decreased notably irrespective of its genomic context. But the quantitative expression of OsEMF2b was depending on the genomic context of the materials being used. When the materials in use were Haitian(2n) and D Haitian(4n) with the same genomic context, the expression level of OsEMF2b dropped markedly whether in the endosperm produced by 2n×4n or 4n×2n in contrast with 2n X 2n. But in the crosses using Nipponbare (2n) and D Haitian(4n) with different genomic context, the expression level of OsEMF2b in endosperm produced by 4n×2n increased dramaticlly compared with the controlled combination.In conclusion, the endosperm development was different mainly in the time of cellularisation after interploidy crosses between 2n and 4n rice; the parental genome dosage could alter the expression pattern of some imprinted genes and the genes which involved in the epigenetic modification also participated in the regulating imprinted gene expression after interploidy crosses.