| Harpin proteins produced in plant pathogenic bacteria can enhance plant growth and increase plant resistance to diseases, insect pests, and envirometal stresses. HpaGxooc, a harpin protein from Xanthomonas oryzae pv. oryzicola, the pathogen that causes bacterial leaf streak in rice. Fragments HpaG1-94, HpaG10-42, and HpaG62-138, contain HpaGXooc regions of amino acid sequence as indicated by the number spans. Here, we reported productivity and biochemical properties of green tea (Camellia sinensis) in response to the fragments and HpaGxooc tested in comparison with an inactive protein control and HpaG1-94. HpaGxooc and HpaG10-42, especially HpaG1-94, have better effects on increasing quantity and improving quality of green tea leaves. In addition, HpaG1-94 and HpaGxooc not only regulate transcriptional expression of genes of growth-related and quality-related, but increase accumulation of catechol in green tea leveas.Riboflavin (VB2) is also a multifunctional elicitor and it plays a key role on growth and disease resistance of man, animals, plants, and microorganisms. Despite essentiality of vitamin B2, however, only plants and certain microorganisms can synthesize it themselves, whereas higher animals including man must obtain it through their diets. Exogenous application of riboflavin can induce ethylene signaling, J A signaling, and defense reaction, which promotes plant growth and increases resistance to disease. But what effects does accumulation of endogenous riboflavin in plant has on plants? It is unknown. And Lumazine synthase which catalyzes the penultimate step of riboflavin biosynthesis is one of key enzymes of riboflavin biosynthesis. Here, we cloned lumzaine synthase gene (OsLS) from rice (oryza sativa) strain Zhonghua11 and transformed it into plant callus cells and transgenic tobacco plants of heterologous expression of OsLS and transgenic rice plants of homologous overexpression of OsLS were gained. Studies on transgenic plants with OsLS gene indicated that the two transgenic plants are similar in accumulation of endogenous riboflavin and disease resistance, but they are different in growth. Moreover, we explored molecular mechanism of growth and disease resistance of the two kind of transgenic plants initially.Mutant is one of most appropriate materials studying Gene functions. And with the rapid development of biological science and technology, the methods of producing mutants are more and more. But, the technique of T-DNA insertion is used widely in plant science because of mature technique, simple operation and convenient analysis. In this study, we gained 121-3 mutant resistant to TMV and ECC by T-DNA insertation and investigated the mutant by molecular biological methods, and eventually, fused sequence of flank plant sequence and border sequence of T-DNA was gained and it was related with plant disease resistance.1. Biological effects of green tea in response to full-length and three functional fragments of HpaGxoocHpaGxooc, a harpin protein elicitor, was produced by a pathogenic bacterium (Xanthomonas oryzae pv. oryzicola) that can cause bacterial leaf streak in rice. To optimize the biofunctions of HpaGXooc, nine functional fragments containing different numbers of amino acid were produced by gene reconstruction and prokaryotic expression in our laboratory. Our previous studies showed that three functional fragments HpaG1-94, HpaG10-42, and HpaG62-138, which contain HpaGXooc regions of amino acid sequence as indicated by the number spans, can enhance growth, increase disease resistance, and induce Hypersensitive cell death (HCD) of tomato and tobacco plants more strongly than HpaGXooc. Here, we report that HpaGxooc and its functional fragments (HpaG1-94, HpaG10-42, HpaG62-138) are applied to green tea(Camellia sinensis), a famous drink plant. And our research results indicate that productivity and biochemical properties of green tea (Camellia sinensis) in response to the three fragments and HpaGxooc are tested in comparison with an inactive protein control and the results show HpaG1-94, HpaGXooc, and HpaG10-42 have better effects on increasing quantity and improving quality of green tea leaves. Especially, compared with the control, the period of picking tea of green tea plants treated with HpaG1-94 and HpaGXooc respectively is 5-16 days earlier, one time more,38.3%and 36.1%increased more in the number of tea buds,50.5%and 37.3%increased more in fresh weight of tea,55.8%and 42.3%increased more in dry weight of tea. In addition, HpaG1-94 can elevate the proportion of tea of high quality to reach 30.8%and prolong tea leaf floating time. Catechol, an antioxidant, which is good to human health, is an important compound which affects tea quality such as colour, aroma, savor, and so on. The content of catechol in green tea is a critical biochemical criterion of the quality of green tea. Our study results indicated HpaGxooc and its three functional fragments all elevated the contents of catechol in green tea, and in contrast to the control, the contents of catechol was added to 64.5%-118.8%. To expatiate on the molecular mechanism of HpaGxooc and its functional fragments affecting green tea, HpaG1-94 and HpaGxooc have many good exhibitions in above research results and therefore the two proteins are selected to carry out the study of gene levels. To reveal the molecular basis for plant growth enhancement, we detected transcriptional expression of tea (Camellia sinensis) expansion-like genes (CsEXPLs) including CsEXPL1, CsEXPL10 and CsEXPL18, which regulate growth of the cell and the plant, and tea quality-related genes including CsCHS, CsDFR, TCS1 and CsSAMS, which were key enzymes of biological synthesis of catethol, anthocyanin, caffeine and theine respectively. The results of our studies indicate that HpaG1-94 and HpaGxooc can elevate transcriptional expression of CsCHS, CsDFR and CsEXPLs in green tea buds, and effect ofHpaG1-94 is much stronger than that of HpaGxooc.Enhancement of expression of CsEXPLs favors increase of biomass of green tea buds, and the induced expression of CsCHS and CsDFR is in favor of the formations of quality of colour, aroma and savor in green tea. However, decrease of expression of TCS1 and CsSAMS in tea buds treated with HpaG1-94 and HpaGxooc is beneficial to control caffeine and theine at moderate and correct levels, which can increase tea quality. Overall, HpaG1-94 much exceeds the full length HapGxooc protein to increase yields and improve the quality of green tea.2. Biological effects of lumazine synthase from rice on plantsRiboflavin (VB2) is also a multifunctional elicitor and it plays a key role on growth and resistant diseases of man, animals, plants, and microorganisms. Despite essentiality of vitamin B2, however, only plants and certain microorganisms can synthesize it, whereas higher animals, including man must obtain it through their diet. Exogenous application of riboflavin can induce ethylene signaling, JA signaling and defense reaction, and enhances plant growth and increase resistance to disease. But what effects does accumulation of endogenous riboflavin in plant has on plant? It is unknown. And Lumazine synthase which catalyzes the penultimate step of riboflavin biosynthesis is one of key enzymes of riboflavin biosynthesis. Here, we cloned lumzaine synthase gene from rice (oryza sativa) strain Zhonghua11,and it was named OsLS. Complete sequence of mRNA of OsLS gene contains 666 base pairs (bp) which encodes a protein containing 221 amino acids, and its amino acid sequence is 68-75% identical with the amino acid sequences of lumazine synthases from higher plants however, OsLS is only 32-49%identical at the amino acid sequence level to its counterparts in microorganisms. A phylogenetic tree is built from the alignment of OsLS and its putative orthologues in several microorganisms and plant species and the result shows OsLS has a much closer relative to those in other higher plants than LSs in microorganisms. OsLS protein with His-tag was expressed in E.coli and Molecular mass of the fusion Protein was predicted to be about 30kD (OsLS, about 23 kD; His-tag, about 7 kD). OsLS protein (100mg/ml) can not elicit hypersensitive response (HR). At the same time, we used localization prediction software, targetp v1.1, to gain the predicted result of OsLS localization, which the reliability of this protein localized in chloroplast is up to 0.879. To test the hypothesis that OsLS is a chloroplast protein, we demonstrated it by determining transgenic tobacco using Western blot and ImmunoGold and determing transgenic rice using ImmunoGold.To understand biological effects of OsLS protein on plants further, OsLS gene was transformated into tobacco (xanthi) and rice (zhonghuall) respectively, and transgenic tobacco plants (LSETT) of heterologous expression of OsLS gene and transgenic rice plants (LSOETR) of homologous overexpression of OsLS gene were gained and these transgenic plants were identified by Southern blot, RT-PCR and Western blot. In addition, transgenic silencing vector of OsLS gene was constructed to transform rice cell, but no transgenic rice plants were produced and this might be as a result that OsLS gene silencing was lethal to plants, which was consistent with previous publications that OsLS gene silencing was lethal to bacteria and fungi and therefore no mutant of OsLS gene silencing was produced. In comparison to wild type tobacco plants, transgenic tobacco plants with OsLS gene have better growth and much stronger disease resistance to tobacco mosaic virus (TMV). Plant growth-related genes such as NtEXPl, NtEXP2 and NtEXP6, and defence response genes such as NPR1, PR-1a, PR-1b and GST1 were determined in LSETT and WT and the results indicated that these genes except NtEXP6, are all induced to express stronger in LSETT, which was consistent with growth and disease resistance of LSETT. Systemic leaves of LSETT and WT were stained with diaminobenzidine (DAB) to determine reactive oxygen species (ROS) in different time points after inoculation with TMV and the experimental result indicated that OsLS had the ability to eliminate ROS produced in plants under the condition of oxygen stress, which could make plant cell to keep away from damage from overoxidation. In recent years, the researchers have a general view that proper concentration of ROS is benefical to induce plant disease resistance and too high concentration of ROS is very harmful to plant. hin1 and hsr203, which are the maker genes of hypersensitive cell death (HCD), were investigated to indicate that their expression levels were also lower in LSETT than in WT, which might have relative to accumulation of ROS in plants. And that we determined the contents of flavins, ethylene (ET) and jasmonates (JAs) in leaves from LSETT and WT respectively, and the results indicated that compared to WT, the levels of the tested substances were all higher in LSETT leaves. Thereinto, Flavins include free riboflavin (vitainm B2, VB2), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and JAs include jasmonic acid (JA) and methyl jasmonate (MeJA). At the same time, these genes, ETR1, CTR1, ERF1, EREBP1 and COI1 in ethylene signaling pathway and jasmonic acid signaling pathway were determined in different tome points postinoculation with TMV and their expression levels were changed, which showed ET and JA signalings were activated much strongly respectively, in LSETT.Research way and method on transgenic rice overexpressing OsLS (LSOETR) were similar with those on transgenic tobacco. Riboflavin, FAD, and FMN were determined in leaves when rice growed to 120d, and in comparison to wild type, the levels of riboflavin, FAD, and FMN in LSOETR were much higher. But LSETR grows much worse and has fewer tillers than the control, and the levels of gene expression of OsMOC1, which controls the number of tiller, and OsGRF1, related to growth, were both weaker than the control, and the heading date of rice of LSOETR was postponed 15d. However, LSOETR growed much higher and its spikes were much longer but its tillers were till fewer than the control after transplanted 157d. Compared to wild type rice, LSOETR behaved much stronger disease resistance after inoculating PXO99 (Xanthomonas oryzae pv.oryzae) and pathogenisis-related genes, OsPR1b and OsPR10 were activated to express much stronger. In comparison to wild type rice, LSOETR can delete accumulation of ROS, the same as LSETT. Clearly, OsLS from rice has multiple biological effects on plant, especially in plant growth and disease resistance.3. Studies on tobacco resistant disease mutantMutant is one of most appropriate materials studying Gene functions. And with the rapid development of biological science and technology, the methods of producing mutants are more and more. In recent years, because method of agrobacterium tumefaciens-mediated transformation is perfect continually, the technique of T-DNA insertion is used widely in plant science in virtue of mature technique, simple operation and convenient analysis. Large numbers of plant mutants were produced by T-DNA insertion and many T-DNA insertion mutant banks have been constructed, and mutants were from arabidopsis, tobacco, rice, and barley. In this study, tobacco (xanthi) cells were transformed by T-DNA insertion of agrobacterium tumefaciens-mediated and 120 transgenic tobacco strains were produced, and these transgenes were inoculated with tobacco mosaic virus (TMV) and Erwinia carotovora pv.carotovora (ECC) respectively, fortunately, we screened a tobacco mutant strain named 121-3 resistant to TMV and ECC. Subsequently, we gained flank plant sequence (281bp) of T-DNA right border by thermal asymmetric interlaced PCR(TAIL-PCR). In wild type tobacco (WT), the flank plant sequence was not transcripted, but it was activated to transcript in 121-3 mutants. And expression of the plant sequence was induced in 121-3 mutants by TMV and ECC, but not in WT. Therefore, we thought that the flank plant sequence was related with disease resistance. Furthermore, both-side sequences of the plant sequence were gained by 3'-race and 5'-race. We found that the flank plant sequence was localizaed at negative strand of T-DNA and partial sequence of negative strand of T-DNA and the flank plant sequence were fused to transcript. In addition, our results indicated that compared to WT, in 121-3 mutant, the levels of JAs and transcription accumulation of ERF1 gene both were increased remarkably, which suggested 121-3 mutant may relate to ethylene signaling and JA pathways. The relations of the flank plant sequence and these signaling pathways and disease resistance are indispensable to study further.4. Conclusive remarks1) HpaG1-94 protein fragment was selected because it not only increased plant yields, but also, most of all, elevated catechol content in green tea.2) OsLS gene was cloned from monocotyledon rice and was transformed into rice and tobacco repectively. Transgenic rice and transgenic tobacco exhibited better resistant to disease, which was a good example in increasing endogenous riboflavin to induce plant defense reaction.3) Tobacco broad-spectrum disease resistant mutant was generated by T-DNA insertion and our studies showed partial sequence of negative strand of T-DNA and the flank plant sequence were fused to transcribe and it may be related with plant disease resistance.
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