Molecular Mapping And Cloning Of Two Rolled Leaf Genes In Rice

Leaves play a very important role in plant development for their function of photosynthesis and transpiration.Leaf morphogenesis,as an important part of plant morphogenesis,determines plant type and agricultural yields.Rice is one of the most important crops in the world,and semi-rolled leaf is one of the most important morphological characters in rice breeding.Therefore,to elucidate the mechanisms of leaf development would be critical not only for us to get a deep understanding of leaf morphogenesis but also for plant type improvement via biodesign.In this study,3 rolled leaf mutants:M425,M429,M475 were isolated from Zhonghua-11(O.sativa ssp.japonica) induced by ⁶⁰Co-y ray.The forth rolled leaf mutant,M7,was derived from japonica variety Nipponbare during the tissue culture process.Of the four mutants,M425 and M7 have the similar phenotype,displaying rolled leaves severely like a cylinder and malformed seeds;the other two mutants, M429 and M475,which look similar to each other too,have a performance of semi-rolled leaf and dense panicle.Anatomical analysis,genetic analysis,gene allelic test and map-based cloning were conducted.The main results were as follows:Part 1:The mapping and cloning of the rice RL9 gene1.Electron microscopic observation revealed that in rl9-1 mutant(M425),the mesophyll cells covered the vascular bundles on the abaxial side of the leaf,where filled with sclerenchyma cells in wild-type;triehoms,which restricted to adaxial domain in wild-type,distributed on both sides in rl9-1;and the arrangement of chloroplast grana lamellae were disordered and irregular in rl9-1. 2.rl9-1 mutant was crossed to the wild-type Zhonghua-11,the F₁ plants were of flat leaves and the ratio of normal plants to mutant plants in F₂ population fitted 3:1, suggesting the phenotype of the rolled-leaf mutant was controlled by single recessive gene.3.Based on the progenies from F₂ and F₃ populations of rl9-1/Dular(O.sativa ssp. indica),the RL9 gene was restricted to a 22-kb region on AP005904 of the long arm of chromosome 9 by using SSR(Simple Sequence Repeat),STS(Sequenced Tagged Site) and CAPS(Cleaved Amplified Polymorphisms) markers.Moreover,rl9-2,the allelic mutant gene of rl9-1,was isolated from M7 by map-based cloning.4.There's only one MYB-like gene located in RL9 anchored region.Sequencing results showed that a 5-bp deletion in exon 1 occurred in rl9-1,which resulted in frame shift;a substitution of A(wild-type) by G at the splice site of intronl/exon2 occurred in rl9-2,which made the intron can not be cleaved accurately and lead to loss-of-function of RL9.5.By means of RT-PCR,coding sequences(CDS) of RL9 gene were redefined.The RL9 gene was located at 103061-108515 of AP005904,consisting of 6 exons and 5 introns,totally 1134-bp CDS,encoding 377 amino acids.6.RL9,the true gene controlled the rolled-leaf phenotype of M425,was confermed by the result of complementation test,which showed that the rl9-1 phenotype was recovered by introduction of the RL9 gene from Nipponbare.7.The results of semi-quantitative RT-PCR showed that the RL9 is expressed in all organs of the wild-type plants,higher in roots,leaves and panicles,and lower in stems and leaf sheathes.8.Sequence analysis revealed that RL9 encodes a GARP domain.9.Alignment of amino acid sequences of RL9 and GARP super-family members was carried out,and a neighbor-joining tree was generated.The results showed that RL9 is homologous to Arabidopsis KANADIs.Based on the role of KANs in Arabidopsis and the performances of loss-fuction of RL9 in rice,it is suggested that RL9 has a function in specifying the abaxial fate of leaves.10.Transient expression analysis using an RL9-GFP fusion protein in onion epidermal cells indicated that the RL9 protein was localized to the nucleus,confirming that the RL9 acts as a transcription factor.Part 2:The mapping and cloning of the rice RL10 gene1.Electron microscopic observation revealed that in rl10-1 mutant(M429),the mesophyll cells covered some vascular bundles on the abaxial side of the leaf,and interestingly,few garland-structures resembling C4 plants existed,in addition, trichoms distributed on both sides.2.rl10-1 mutant was crossed to the wild-type Zhonghua-11,the F₁ plants were of flat leaves and the ratio of normal plants to mutant plants in F₂ population fitted 3:1, suggesting the phenotype of rl10-1 mutant was controlled by single recessive gene.3.Using the progenies from F₂ and F₃ populations of rl10-1/Dular,the RL10 gene was restricted to a 38-kb region between markers f70 and f87.Moreover,rl10-2,the allelic mutant gene of rl10-1,was isolated from M475 by map-based cloning.4.Five genes located within the RL10 anchored region.Sequencing results of the five genes showed that gene 2 mutated in both two rl10 mutants,i.e.a substitution of T (wild-type) by C occurred in rl10-1,which resulted in a Ser transformed to Pro;a 2-bp deletion occurred ahead of the stop codon in rl10-2,which lead to post-termination of translation.5.RNAi aimed at the candidate gene 2 in wild type plants could result in the mutation phenotype of rl10 mutants.6.The results of semi-quantitative RT-PCR showed that the RL10 is expressed in all organs of the wild-type plants,higher in leaves,leaf sheathes and panicles,and lower in roots and stems.