水稻突变群体的构建及功能基因组学

随着水稻基因组全序列的测定完成，功能基因组学已成为重点研究内容。功能基因组学主要研究生物有机体内各基因的生物学功能进而了解所有基因如何协调发挥作用完成一系列的生长发育过程。目前已经发展了多种分析鉴定基因功能的方法，其中最直接最有效的方法是构建饱和的基因突变群体，通过突变体分析鉴定基因功能。本文主要阐述了各种构建方法及其优缺点以及在功能基因分离鉴定上的应用。自发突变的频率极低，且自发突变基因的分离难度比较大，只能作为突变群体构建的辅助方法。利用EMS等化学诱变剂可以在短时间内构建大量点突变群体，并可用TILLING进行突变检测，但多位点的点突变使突变表型难以鉴定。由快中子等物理诱变也可以在短时间内构建大量缺失突变体，且可用Ddeteagene系统进行检测；但多基因缺失、多位点缺失和内含子缺失等使突变表型的分析可能无法进行。利用T—DNA、转座子和反转录转座子等构建插入突变体已经成为突变库构建的主要方法。T—DNA插入已成功应用于水稻大规模突变体的构建，但只限于转基因效率较高的品种；T—DNA在基因组中整合的复杂性以及转基因过程中由组织培养等引发的突变等，增加了突变体表型和分子分析的难度。Tos17是目前应用最为成功的反转录转座子，但多拷贝的插入使突变体的表型鉴定和分子鉴定较为困难，因为只有10％左右的突变性状是由Tos17插入引起的。理论上，Ac／Ds双因子系统是目前最理想的水稻插入突变库构建体系，Ds的单拷贝插入，大大方便了突变体的表型分析和分子鉴定；Ds的回复突变，可以验证突变表型是否由Ds插入引起；但在血转座酶驱动下Ds可能发生的多次跳动所形成的痕迹(footprint)也可能影响突变表型的分析。RNAi可以有效地使目标基因沉默，但并不是所有基因均可被RNAi沉默；对多因一效基因或同源性较高的基因家族，RNAi会同时作用这些基因，沉默表型很难鉴定。可见，每一种方法都有各自的优缺点，但不同的方法是可以互补的，通过各种方法是能够构建成理想的水稻突变库的。

Rice Mutant Population and its Applications on Functional Genomics

With the completion of the rice genome sequence to assign a function to unknown or predicted gene becomes a major task in functional genomics. Functional genomics is a general approach toward understanding how the genes of an organism work together by assigning new functions to unknown genes. Now so many new technologies have been developed to exploit gene functions. Among them is to identify all genes by mutant analysis based on a saturated mutant population, which is a straight and efficient way to understand the roles of all genes. In this paper, various methods to produce mutants and their applications on functional genomics were reviewed and commented. Spontaneous mutants can be used only as a supplemental resource because of its low frequency of mutation and its difficulty in identifying mutated genes. Some chemical mutagenesis such as EMS can be efficiently used to produce a large number of point mutants in a short period and the induced mutants can be detected by TILLING. But it is difficult to detect a point mutation and its phenotype when there are multiple point mutations in one mutant. Similarly, Fast neutron can be applied for mutagenesis and producing a large number of deletion-based mutants in a short period, and the deletion mutants can be screened by Deleteagene. However, it is also very difficult to identify a deletion mutant and its phenotype when the induced deletion occurs covering multiple genes or within an intron. Insertional mutagenesis based on T-DNAs, transposons and retrotransposons is becoming a major approach to produce a saturated mutant pool. A large number of T-DNA insertion lines have been produced in rice; but T-DNA insertional mutagenesis can be used only for those rice varieties with highly efficient transformation and it is difficult to analyze an insertion line because of complex patterns of T-DNA integration into the rice genome and mixture with mutations induced from tissue culture. The retrotransposon Tos17 has been successfully applied for rice functional genomics. But it is also difficult to identify a mutant related to Tos17 because there are multiple copies of Tos17 in a mutant and only about 10 percent of mutants are from Tos17 insertion. Theoretically, Ac/DS two-element system is regarded as a best approach for rice insertional mutagenesis. It is very easy to identify a mutant because of single copy of Ds insertion. An additional advantage is that Ds can be excised from disrupted gene in the presence of Ac transposase, resulting in phenotypic reversion to the wild type or giving rise to alleles with weaker phenotypes. However, it is also difficult to identify a mutant when there are Ds excision footprints in the mutant caused by Ds multiple excision-insertion events in the presence of Ac transposase. RNAi can efficiently silence a gene, but not all genes can be silenced. In addition, RNAi can interfere in genes with redundant and overlapping functions or gene families with high homolog in sequence, making it difficult identify a silenced gene. So it is obvious that each method has its advantage and disadvantage and different methods should be combined to produce a saturated mutant population.