Cloning And Functional Analysis Of Rice Lipoxygenase-Related Genes And Its Application In Rice Breeding

Lipoxygenase(EC:1.13.11.12,referred as LOXs),a large family of non-heme iron containing dioxygenases.They catalyze the insertion of molecular oxygen into PUFAs that contain one or more(1Z,4Z)-pentadiene moieties to yield the corresponding(1S,2E,4Z)-hydroperoxides.In plants,LOXs mainly catalyzes linoleic acid(LA)and linolenic acid(Le A),and the catalytic oxidation of C9 and C13 corresponds to the generation of 9-HPOS and 13-HPOS.Therefore,LOXs is divided into two subtypes,9-LOXS and 13-LOXS.In animals,LOXs mainly catalyzes arachidonic acid,and the oxygenation site is mainly in C5,C12,C15,and participates in inflammation and other physiological processes.LOXs occur ubiquitously in various organs and tissues of plants,and also plays an important role in the process of biotic and abiotic stress.Lipoxygenase exists widely in animals,plants and microorganisms.Plant LOXs is mainly located in chloroplast,vacuole and cytoplasm,which is usually in a resting state and starts only when the organism develops to a certain stage or is subjected to environmental stress.Studies have conf-irmed that LOXs may provide precursor substances needed for the synth-esis of JA(JA),methyl ester and guaiacin,and play a role in the formation of fruit flavor substances and insect and disease resistance pathways in plants.The rice lipoxygenase family consists of 13 members,of which 6belong to the 9-LOXS subtype,7 belong to the 13-LOXS subtype.In the previous work of the laboratory,transcriptomics sequencing analysis was carried out on the samples obtained by Magnaporthe Grisea inoculation experiment of japonica rice variety ’Yun yin’ at seedling stage,and the candidate gene Os02g0194700(13-LOX)with significantly different expression was obtained.In this study,Os02g0194700 was taken as the research object,and Yun yin and Nipponbare were used as background materials to explore the potential mechanism of jasmonate biosynthesis pathway,biotic and abiotic stress pathway.The specific results are as follows:1.The gene was successfully cloned from ’Yunyin’ with the sequence of Nipponbare genome as a reference.The promoter sequence of the gene was analyzed by bioinformatics technology,and it was found that there were several regulatory elements responding to stress in the promoter sequence of the gene,which suggested that it might play a role in biotic and abiotic stress.2.We analyzed the LOX gene family and molecular weight and isoelectric point of corresponding proteins were counted.The phylogenetic tree of rice LOXs family genes was constructed,and the evolutionary relationship of LOXs family members was analyzed.3.CRISPR-Cas9 knockout vector,overexpression vector,subcellular localization vector and tissue specific expression vector of Os02g0194700 were constructed.In order to avoid gene redundancy,multiple gene knockout vectors were constructed,and T0 generation positive plants were obtained.The results of subcellular localization showed that the gene was mainly located in chloroplasts.GUS staining of transgenic plants showed that this gene was highly expressed in leaves and leaf sheaths,while the expression level was low in stems and roots.4.In this study,real-time fluorescence quantitative PCR was used to analyze the expression pattern of rice LOXs family genes under blast stress.The results showed that Os02g0194700,Os03g0699700,Os03g0700400,Os03g0700700,Os05g0304600 and Os08g0508800 were induced by Magnaporthe grisea.These two genes may play a role in rice defense against Magnaporthe grisea.Lipoxygenase activity was significantly inhibited after treatment with lipid-oxygenase inhibitor NDGA and catechin.5.Five proteins that may interact with Os02g0194700 were screened by yeast two-hybrid system.And verified by Bi FC and Pull down tests.