Screening And Characterization Of The OsMAP65 Family T-DNA Insertion Mutants In Rice

Microtubule (MT) is one of the main constituent elements of the cytoskeleton, the basic unit of which is tubulin. MT has the dynamic instability and be involved in many cellular life processes. In order to respond to a wide variety of internal and external stimuli in cell, tubulin plays an important role in abiotic stress tolerance (high salt, hypertonic, disease, etc.) with the assistance of the microtubule-associated protein (MAPs). The abnormal expression or modifi-cation of MAPs may lead to microtubules disorder and diseases, such as cancer. MAP65 is a specific microtubule-associated protein in plant. Recent studies have shown that the knockout of MAP65-1 in salt stress can lead to microtubule depolymerization, and the over-expression of MAP65-1 would increase salt tolerance in Arabidopsis thaliana.These results, indicate that MAP65 has a major impact on microtubule organization when the plant is subjected to various abiotic stress. In plants, the research of AtMAP65 genes family have been reported, but the research of OsMAP65 family in the mode monocot-rice (Oryza sativa) is still very lacking. Therefore,screening of T-DNA insertion mutants in the OsMAP65 family help better under-standing molecular mechanism underlying stress tolerance in rice.In this study, following experiments were already carried out:(1) analyzing the different parts expression of the OsMAP65 family members under adversity stress in Japonica Varieties (Nipponbare) by the semi-quantitative RT-PCR; (2) screening and characterization for T-DNA insertion homozygous mutants of the OsMAP65 related-gene in rice; (3)characterization of homozygous mutant phenotypic and agronomic traits;(4)the identification of cell death, inoculation test and Tail-PCR analysis in OsMAP65-1 homozygous mutant map65-1. The results are shown as follows:(1) Under high salt and drought stress, the different parts expression of the OsMAP65 family members in Japonica Varieties (Nipponbare) are not the same. For example,after the treatment of salt and PEG6000,the expression of OsMAP65-1 is different.The overground part of rice didn’t express at Oh under the salt treatment, but generated induced expression after 3h, and continued to 6h, and the express quantity had continuously decreased after 9h.The overground part of rice didn’t express under the drought stress,and the underground part has been expressed,which indicating that these stress treatment had an impact on the gene.(2) To identify 11 kinds of T-DNA insertion mutants involved in OsMAP65 related-gene, purchased from abroad by using "double primers method LP+RP, RP+RB)" and "three primers method (LP+RP+RB)".The results of identification:there are three homozygous mutant lines,namely, map65-1,map65-3,map65-7:and there are four heterozygous mutant lines, namely, map65-2, map65-6,map65-8, map65-10.(3) During the seedling stage, compared to wild type japonica Dongjin, T-DNA insertion homozygous mutant map65-1 and map65-3 grow slower and dwarfish; compared to wild type, mutants have more tillers and look more compact after being planted in the field; although mutants have more effective panicle than wild-type during the mature stage, the full real rate of them are very low, almost of them are shriveled grain. In addition, it was found that the leaf of OsMAP65-1 homozygous mutant map65-1 appeared reddish-brown spots from five leaves stage to mature stage, which is suspected to be lesions in the whole growth stages.Agronomic traits identification results show that compared with wild type, the T-DNA insertion homozygous mutants have a highly significant increase on tiller number, effective panicle, spike length of main axis; the homozygous mutants have a decrease on secondary branch number per panicle, and have a highly significant decrease on plant height, panicle exsertion, grain number per spike, sword leaf length, thousand grain weight. However, the homozygous mutants have no significant differences in the grain length, grain width, grain thickness and sword leaf width.In stipes, the first stipe is significantly decrease, and the other stipes of map65-1 are all highly significant decrease.(4) we have proved that the cells of lesion leaf of the OsMAP65-1 homozygous mutant map65-1 are dying by utilizing NBT, DAB staining. We also found that the plant has certain resistance to P2 (PXO86), which is one of the physiological races of XOO. Besides, we detect the T-DNA fragment insertion number and insertion position of the homozygous lines of map65-1 by Tail-PCR.