Fine Mapping And Cloning Of DNL1 And RL3(t) Genes, Responsible For Leaf Shape Formation In Rice

The morphology of rice leaf is one of the major components of the ideotype in rice, it is closely related to the photosynthesis efficiency, and consequently contributing to the yield formation. Rice is a crop of polymorphism, and its leaf shape has magnificant diversity. Generally, the common cultivated varieties displayed flat and wide leaves, however a few varieties displayed rolled or narrow leaves. Up to date, although a few genes involved in rice leaf development have been identified and functionally analyzed, the knowledge accumulated about the genetic mechanism of rice leaf development is still unclear yet. Consequently, to elucidate its molecular mechanism, is not only importent to our theoretical understanding but also significant to practical application.In the present study, three mutants derived from the indica cultivar 93-11 via the radiation of 60Co-y ray were temporally designated as dnll, rl3(t)-1 and rl3(t)-2. The dnll mutant displayed dynamic narrow leaves; the phenotype of rl3(t)-l was similar to rl3(t)-2, both of which had inner curled leaves. In the present study, morphological observation, genetic analysis of mutant traits and map-based cloning have been conducted. Moreover, we tested the allelomorphism of rl3(t)-1 and rl3(t)-2, and identified the function of DNL1.Part1. The cloning and functional identification of DNL1 gene1. A dynamic narrow leaf mutant, initially named dynamic narrow leaf 1 (dnll) was obtained via the radiation of 60Co-y ray. Characterization of dnllmutant showed although the leaf widths of dnll/Osiaa6 were similar to those of the WT at the seedling stage, the leaf widths became narrow since the 10th leaf emerged in dnll. Interestingly, the leaves which appeared after the 14th leaf restored to normal again. Another typical characteristic of the dnll/Osiaa6 mutant is that the mutant has no root hairs. Moreover, some other agronomic traits of the mutant were also quite different from those of the WT control. In addition, it was found that the sawtooth hairs at the leaf margins were also poorly developed.2. An F2 segregating population was generated by crossing dnll mutant with its wild type 93-11. The Fis showed normal leaf widths, and in the F2 population, the ratio between plants with normal leaves to those with dynamic narrow leaves well fit the Mendel’s ratio (3:1), strongly indicating that the dnll mutant was governed by a recessive nuclear gene.Using the F2 and F3 population derived from crosses between dnl1/0siaa6 mutant and WYJ8, WYJ7 (japonica cultivar) respectively, DNL1 gene was located between AP002972 and AP003768 on chromosome 1, about 20kb, by using SSR、STS and CAPS markers.3. There is only one predicted open reading frame (ORF), encoding an Aux/IAA protein, named as OsLAA6 by Jain et al. DNA sequencing of the gene in the mutant showed the following changes, three single base substitutions in intron 2, one A to G synonymous mutation in exon 4, one two-bp insertion and one G to C substitution in the promoter.The phenotypes of the mutant, were rescued when the normal OsLAA6 gene was introduced into the dnl1 mutant. This results strongly demonstrated that OsIAA6 is the candidate of DNL1 gene.4. The results of qRT-PCR revealed that DNL1/OsIAA6 expressed in all organs investigated, such as roots, culms, leaves, leaf sheaths, and young panicles. Furthermore results of GUS staining showed that DNL1/OsLAA6 constitutively expressed in all the tested tissues or organs, especially in the roots and root hairs.As to DML1/OsIAA6-GFP fusion protein, fluorescence was observed solely in the nucleus, indicating that DNL1/OsIAA6 localized in the nucleus.5. DNL1/OsIAA6 is sensitive to temperature and the rice leaves would turn narrow if its expression does not reach a certain high level in the high temperature.6. Treat the mutant dnl1 and WT with different concentrations of NAA, and the results confirmed that the mutant was less sensitive to the inhibitory effect of high concentrations of auxin. By adding low concentrations of NAA in the medium, the phenotype of none of root hair of the mutant can be rescued.7. Artificial down-regulation of DNL1/OsIAA6 resulted in appearance of dynamic narrow leaf and blocking of root hair elongation, and it is similar to dnll mutant.8. OsZHD1、 ADL1、SRL1 and NRL1 genes were responsible for leaf shape formation, and it showed that when the expression of OsZHD1 turned higher, it may lead to the phentype of rolled leaves. Meanwhile when the expression of ADL1, SRL1 and NRL1 became lower, it contributed to appearance of rolled or narrow leaves. In the present study, the expression of ADL1, SRL1 and NRL1 turned lower in dnl1 compared with WT, and the expression of OsZHD1 gene was higher in dnl1, It is suggested that when the expression of DNL1/OsIAA6 became lower, the expression of these genes changed implying that these genes may function in the down-stream of DNL1/OsIAA6 gene. Similaly, it was shown that OsAPY gene was involved in root development, when the expression of OsAPY turned lower, the root hair elongation was blocked. In the mutant dul1, the expession of OsAPY also came lower, suggesting that OsAPY functions in the down-stream of DNLl1/OsIAA6, too.Part2. Fine mapping of RLS(t) gene1. Morphological analysis showed that, compared to the wild type, the two mutant rl3(t)-1 and rl3(t)-2 exhibited typically incurved leaves, reduced plant height, shorter panicle length and lower fertility.The result of cytological analysis indicates that the change of the morphology of the bulliform cells may be the reason causing the adaxial rolled leaves.2. Two F2 segregation populations were generated by crossing two mutants with a japonica cultivar WYJ8 and its wild type 93-11, respectively. The F1s have normal leaves,χ2-test showed the segregation for leaf shape in F2 is significantly distorted from 3:1 Mendel’s ratio, with severely fewer adaxially rolled leaf plants. Crossing between the two mutants, the F1 (rl3(t)-1/rl3(t)-2) plants exhibit rolled leaf, suggesting they are allelic.lt is deduced that rolled leaf mutant is possibly controlled by a recessive gene.3. Using the F2 and F3 population (rl3(t)-11 WYJ8 and rl3(t)-2/ WYJ8, RL3(t) gene was successfully assigned to a region governed by S3-36 and SAL1, with about 23kb-long DNA fragment.4. In the target region, there are four open reading frames (ORFs) in total. It has been found that one ORF was predicted to encode a protein containing the F-box domain. In addition, one zinc finger protein gene, one amylase gene as well as one DVR gene are also been predicted.