The Three Resistance Signaling Pathway-related Genes In Transgenic Tobacco Expressing Hpa1_Xoo-N21

Harpins are a class of helper proteins secreted by the type Ⅲ secretion systems of many Gram-negative phytopathogenic bacteria and a class of non-specific elicitors that elicit a hypersensitive response (HR) in nonhost tobacco, induce disease and insect resistance in plants, and promote plant growth. Hpa1Xoois encoded by hpa1Xoo gene from the rice blight bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). The Hpa1Xoo N-terminal coiled-coil (CC) of Xoo can elicit HR in tobacco leaves, and is an important functional and structural unit. In order to clarify N21 function and mechanism, this study intends to determine the transcriptome profiles of transgenic N21 gene and related genes (hpa1Xoo and AB) tobaccoes using DNA microarray technique and investigate the expressions of salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) resistance signaling pathway genes and defense-related genes.This paper detected the expressions of 3 desease resistance signaling pathway genes and defense-related genes in transgenic tobacco expressing hpa1Xoo、AB、N21, and parents (CK) tobacco, using microarray analysis technology. According to the results of the microarray analysis, the 2-fold (ratio>2 or ratio<0.5) differential expression was selected as the standard. When transgenic hpa1Xoo tobacco compared with CK tobacco, there were 4528 differentially expressed genes in total, in which 2237 genes were up-regulated and 2291 genes were down-regulated. Contrasting transgenic AB tobacco and CK tobacco, there were 1261 up-regulated genes and 999 down-regulated genes. There were 1204 up-regulated genes and 1362 down-regulated genes in transgenic tobacco expressing N21 when contrasting with CK tobacco. Being compared with transgenic N21 tobacco, transgenic hpa1Xoo tobacco had 4273 differentially expressed genes in total, in which 2330 genes were up-regulated and 1943 were down-regulated. Contrasting transgenic AB with N21 tobacco, there were 1442 up-regulated genes and 1838 down-regulated genes. Compared to transgenic AB tobacco, there were 1017 up-regulated genes and 805 down-regulated genes in transgenic tobacco expressing hpa1Xoo. These differentially expressed genes are related to plant stress responses, growth and development, lipid metabolism and the SA, JA and ET signaling transduction pathway and defense responses.Further study revealed that, (1) there were 7 down-regulated genes, AT3G28910, AT2G46370, AT3G09600, AT4G35770, AT4G09460, AT2G40000 and AT5G44030, related to the JA signaling transduction pathway in transgenic N21 tobacco compared with CK tobacco. AT4G35770 encodes SEN1 and is related to senescence, JA and wound response, and also involved in oxidative stress response. (2) AT1G28380, AT4G09460, AT2G40000, AT5G42950, AT4G05320, AT1G13740, AT1G64160, AT5G48930 and AT4G27430 are all related to SA resistance signaling transduction pathway. AT5G48930 and AT4G27430 were up-regulated expression, the others were all down-regulated. AT1G28380 was predicted to encode a protein NSL1 involved in negatively regulating SA-related defense responses and cell death programs. AT4G05320 is gene UBQIO, and encode the highly conserved 76-amino acid protein ubiquitin induced by SA but independent of NPR1. (3) AT2G46370, AT3G28910, AT3G09600, AT5G25190, AT2G40000 and AT5G44030 are all related to ET signaling transduction pathway. They were all down-regulated genes. AT5G25190 is an ethylene-responsive transcription factor ERF003, encoding a member of ERF/AP2 transcription factor family ERF (ethylene-responsive factor) subtribe B-6. (4) And there were some defense response genes, AT1G58848, AT5G48930 and AT1G72870, are related to apoptosis, PCD and lignin biosynthesis respectively. AT1G58848 is up-regulated, and encodes a putative disease resistance protein RDL6/RF9. AT5G48930 is up-regulated, related to flavonoids biosynthesis and lignin biosynthesis. It encodes a hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT). HCT is highly induced by different abiotic stress, such as mechanical injury, H2O2, cold, ABA, SA and drought. AT1G72870 encodes TIR-NBS class of disease resistance protein.It was observed that many differentially expressed genes are related to more than one resistance signaling pathway. AT2G46370 and AT4G09460 are not only related to JA but also related to ET and SA signaling pathway respectively. In addition, AT3G28910, AT3G09600, AT2G40000 and AT5G44030 are all related to the three resistance signaling pathways. AT3G28910 is jasmonic acid-amido synthetase JAR1, and JAR1 catalyzes the formation of a biologically active JA-Ile conjugate. JA-Ile promotes the interaction between JAZ1 and COI1 in JA signaling pathway. JAR1 also plays an important role in ET signaling pathway. AT4G09460 is transcription repressor MYB6, located in the nucleus, and respond to JA and SA. AT3G28910 is myb domain protein myb30, related to plant HR and respond to SA、JA and ET. AT3G09600 is not only participating in 3 signaling pathway, but also related to abscisic acid、gibberellin and auximone responses. AT2G40000 encodes nematode resistance protein HSPRO2, which positively regulates basal defense response and acts on SA downstream, and negatively regulates it through JA and ET. AT5G44030 is cellulose synthase CESA4, related to the plant resistance response to fungus and bacterium.Using EF-1α as the internal reference gene, this study verified some genes related to SA, JA and ET signaling transduction pathways in transgenic hpa1Xoo-N21 gene tobacco compared with CK tobacco by fluorescence quantitative RT-PCR. The results indicated that the 3 signaling pathway genes, such as AT2G46370, AT3G09600 and AT5G25190, were expressed, and can also detect the expression of other important the defense-related genes AT1G58848, AT5G48930 and AT1G72870, were also expressed. The verified genes were consistent with the results of gene chip analysis in general.