水稻二倍体和四倍体杂交的胚乳发育和印记基因分析

Genomic imprinting refers to an epigenetic mechanism by which the selective expression of a parental allele dependents on its parent of origin. Imprinting primarily occurs in the endosperm, a triploid nutritious tissue of flowering plants. In Arabidopsis, imprinting is thought to regulate the timing of cellularisation of endosperm to influence seed size. Rice is one of the most important food crops. Endosperm is the major source for food. It is important to understand the role of imprinted genes in regulating endosperm development. Using the seeds with distinct phenotypes from the reciprocal crosses of diploid (2n) and tetraploid (4n) rice, we will investigate the endosperm and embryo development cytologicaly to understand if imbalanced contributions of maternal and paternal imprinted genes result in different endosperm phenotypes between the 2n and 4n reciprocal crosses. We will also analyse the expression level and imprinting mode of previously identified imprinted genes in the parental genome imbalanced crosses. Seeds from interploidy crosses will be rescued on medium formulated for cereal early seeds by adding DNA methylation inhibitor (5-Azacytidine). This study will illustrate that how expression of imprinted genes is affected and, at to what extent, imprinting will be deregulated in interploidy crosses. Thus, this project will test the widely accepted parental conflict theory and provide new insight about ?the mechanism and function of imprinting.

基因印记是指子代仅表达亲本之一基因拷贝的现象。植物中，印记主要发生在三倍体胚乳中。拟南芥中，印记通过控制胚乳细胞化的时机来调节胚乳发育进程。水稻是重要的粮食作物，胚乳亦是人类食用的主要部分，探索印记基因对胚乳发育的作用显得尤为重要。本研究根据水稻二倍体（2n）和四倍体（4n）正、反交后种子发育存在明显差异且败育这一现象，以前期研究确定的100多个新的印记基因为基础，通过胚乳发育观察、印记基因表达测序、添加DNA甲基化抑制剂等方法，重点研究印记基因在父母本基因组剂量失衡下的表达量变化和印记方式变化，以及降低甲基化水平能否改善胚乳败育。本结果将有助于确定种子败育的原因及方式，印记基因表达与胚乳败育之间的关系，从而为减少异交败育或促进胚乳发育提供参考；也可进一步验证"亲本冲突学说"在水稻中的适用性，为揭示水稻等禾谷类作物印记调控模式奠定重要基础。

在不同倍性水平的水稻之间的生殖隔离表现为胚乳和胚胎在通过倍性交叉产生的种子败育。 基因组印记被认为是建立后合子杂交屏障的基础机制。我们研究发现二倍体粳稻和四倍体籼稻之间的正反交后可导致种子发育终止。. 来自这些杂交的三倍体种子已经中止发育，并且不能在土壤中发芽，但是可以在培养基中被拯救而成苗。与先前的发现一致，2n×4n杂交，产生的后代具有过量的父本基因组，从而产生更大的胚胎；而4n×2n（♀-♂）的后代则具有过量母体基因组，容易发生早期胚乳细胞化。观察父本剂量过剩的杂交种子中，没有发生细胞化，球形胚发育终止，说明在胚乳发育中，胚乳细胞化是非常重要的。此外，我们在父本剂量过剩的杂交后代的果皮实质细胞中，发现淀粉颗粒持续沉积，而在母体剂量过剩的杂交后代的胚乳发育中，果皮淀粉逐渐减少并重新定位于平衡。细胞化和淀粉沉积发生在胚乳，表明父母基因组平衡影响通过细胞化和淀粉沉积的果皮淀粉重定位。我们还观察印记基因和表观遗传调节因子OsFIE2和OsMET1b的表达的改变，说明印记丢失，这意味着印记和表观遗传机制在调节不同亲代基因组剂量对胚乳发育的作用中有潜在作用。. 我们的结果支持母体基因组剂量促进胚乳细胞化和父系基因组剂量通过贡献不同组的印记基因延迟或抑制细胞化的假说。