生菜结球控制基因LHL1和LHL2的遗传克隆及其作用机理

Lettuce (Lactuca sativa) is one of the most important vegetables worldwide. Due to its short generation life, easy management, completed genome sequence and high transformation efficiency, lettuce has become a model species for the Compositeae family. More than half of leafy lettuce in the world is iceberg lettuce (head lettuce), and iceberg lettuce has become more and more popular in Chinese consumers. In our preliminary study, a F2 population was derived from a cross between an iceberg lettuce (heading) and a Romaine type lettuce (no heading). In the segregating F2, the leaves of 50 individuals with the best head were pooled and the 50 individuals with no head were pooled. RNAs were extracted from the two pools and sequenced. Data analysis identified two loci associated with heading. F2 individuals with one of the detected loci being heterozygous and the other locus being homozygous were chosen for further analysis. Seeds were harvested from those individuals (F2:3). In a F2:3population with obvious Mendelian ration (3:1), the target locus is linked with the heading trait, and therefore we successfully constructed a single gene segregating population for the heading trait. In this proposed study, we will fine map the gene and clone it using map-based cloning strategy. Then, we will study its molecular mechanism on generating lettuce head. At the same time, we will map and clone the second gene using similar approach. The proposed study will help us understand the mechanism of heading, which will facilitate future breeding of iceberg lettuce. Furthermore, the results may be applied to the studies and breeding of vegetable crops with heading phenotype, such as endive, cabbage, brussel sprout, and Chinese cabbage.

生菜是世界上最重要的蔬菜之一，在我国也越来越受消费者喜爱。结球生菜占世界生菜总量的一半以上。本课题前期通过结球生菜与罗马生菜杂交获得结球性状连续分布的F2分离群体。对F2群体的两个极端池（结球和不结球）进行RNA测序，鉴定出2个控制生菜结球性的主效QTL位点，命名为LHL1（Lettuce Head Locus1, LHL1）和LHL2。为克隆主效QTL，成功构建了一个F2:3群体，即LHL2为杂合而LHL1为纯合的F2植株进行自交，获得单基因分离的F2:3群体。利用该群体对LHL2进行了初定位，该基因与7号染色体24Mb处的标记共分离。本研究拟利用该单基因分离群体对LHL2进行精细定位并进行图位克隆，进而分析其作用机理。同时，我们将利用相同方法克隆另一个结球控制基因LHL1。本研究将揭示生菜结球形成的分子机理，对培育优良的结球生菜并对其它结球蔬菜的研究具有重要的指导意义。

生菜是世界上最重要的蔬菜之一，在我国也越来越受消费者喜爱。结球生菜占世界生菜总量的一半以上。本项目通过结球生菜与罗马生菜杂交获得结球性状连续分布的F2分离群体。对F2群体的两个极端池（结球和不结球）进行RNA测序，鉴定出2个控制生菜结球性的主效QTL位点，命名为LHL1（Lettuce Head Locus1, LHL1）和LHL2。为克隆主效QTL，成功构建了一个F2:3群体，即LHL2为杂合而LHL1为纯合的F2植株进行自交，获得单基因分离的F2:3群体。利用该单基因分离群体对LHL2进行精细定位并进行图位克隆，确定LsSTM2为控制结球形成的候选基因，通过转基因验证了该基因功能。进一步分析了其作用机理，当LsSTM2有转座子插入时，其表达量上升。由于LsSTM2基因的表达量上升促进了生菜结球的形成，而LsSTM2表达量的的改变是由于在结球生菜中该基因启动子区有一个转座子的插入，该转座子的插入改变了LsSTM2的表达模式。另外，LsSTM2通过结合改变叶片背腹性基因LsAS1的表达导致生菜叶片背腹性的改变从而促进结球的形成。本研究揭示生菜结球形成的分子机理，对培育优良的结球生菜并对其它结球蔬菜的研究具有重要的指导意义。