Detection Of Hpa1_Xm-transgenic Tobacco Plants And Interaction Between IAA And Harpin_Xm Protein

In gram-negative bacteria, hrp genes encoded a kind of protein Elicitors named Harpin, which secreted by type ? secretion system.It can make the non-host plants bring about HR(hypersensitive response) reaction.Exogenous Harpin protein can cause the plant to produce systemic acquired resistance, and stimulate plant a variety of beneficial reactions.Hpa1Xm,identified from Xanthomonas citri subsp.malvacearum,can encode harpin protein with common harpin characteristics of glycine-rich and heat-stable.The forecast of secondary structure revealed that 1-15 amino acids may be a potential signal peptide analogs on the N side.We transferred hpaXm and hpaXm?LP into tobacco in previous study.In order to study the HpaXm and the expression and function of HpaXm influenced by the putative signal peptide,we delved the genetic stability of transgenic tobacco and endogenous expression harpin protein.It provided theoretical guidance and technical basis for the efficient development and application of hpaxm.The main results were as follows:1.We analyzed biological and biochemical characters in hpaXm-transgentic tobacco.hpaXm ?LP-transgentic tobacco?non-transgentic tobacco and transgentic tobacco in different progeny.Kanamycin resistance tests,PCR,RT-PCR and PCR Southern blot analysis were uesd to screen and identify Ti to T3 progeny of hpaXm-transgentic tobacco and hpaXm?LP-transgentic tobacco lines.Using PCR and PT-PCR method,the genomic intergrantion and expression of the transferred gene can be proved. Biological characters of seedling stage tobacco experiment shows that there was no significant difference between hpaXm-transgentic tobacco and hpaXm ?LP-transgentic tobacco,even the numerical value of hpaXm-transgentic tobacco is slightly higher than hpaXm?LP-transgentic tobacco.Determination of chlorophyll in transgenic tobacco was higher than wild type plants.And the total chlorophyll content of hpaXm-transgentic tobacco was higher than hpaXm?LP-transgentic tobacco.Determination of the activity of defense enzymes indicated that transgenic tobacco plants are higher than wild type plants in PAL, POD and PPO activity .At the same time,hpaXm-transgentic tobacco was higher than hpaXm?LP-transgentic tobacco in PAL and POD activity.Determination of physiological and biochemical indexes in the same variety of transgenic tobacco plants indicated that the numerical investigation of T2 generation was slightly higher than T3 generation.The determination results of DAB staining and H2O2 content showed that transgenic tobacco plants were higher than wild type plants in H2O2 content.Besides,the H2O2 content of hpaXm-transgentic tobacco was slightly higher than hpaXm?LP-transgentictobacco.Treated with trypan blue,micro-HR emerged on transgenic tobacco leaves. In the same perspective, there was no significant difference in the number and color of cell clusters between different transgenic tobacco and same variety transgenic plants of T2 and T3.It showed that hpaXm? hpaXm?LP gene had indeed been stably integrated into the transformed tobacco genome, and it can expression stably and efficiently.2.We tested 10-4 mol/L IAA effect on the expression of endogenous HpaXm? HpaXm?LP.At the same time,we probed the regulation of 10-4 mol/L IAA on the expression of endogenous HpaXm?HpaXm ?LP.Transgenic tobacco leaves were treated with 10-4 mol/L IAA and 500?M TIBA in this article.Determination of oxygen burst and micro-HR showed that 10-4 mol/L IAA had an inhibitory effect on oxygen burst phenomenon and micro-HR induced by endogenous hpaXm and hpaXm?LP.Meanwhile, 500 ?M TIBA can inhibit the effect of IAA.Real-time qRT-PCR was used to evaluate the expression of hsr203J,chia5,aoxl and IAA9 genes in tobacco leaves treated with IAA or IAA combined with TIBA at 1, 3, 6, 9 and 12 h.The expressions of hsr203J and aoxl in hpaXm-transgentic tobacco were similar to hpaXm?LP-transgentic tobacco.Both of them showed that the expressions of hsr203J and aoxl following the treatment of the interaction of IAA and TIBA was higher than the treatment of IAA. For the rest,the expressions of chia5 gene between hpaXm-transgentic tobacco and hpaXm ?LP-troansgentic tobacco was well out of line with IAA9 gene. Result of qRT-PCR revealed endogenous expression harpin protein could regulate the expression of hsr203J,chia5,aox1 and IAA9 genes.And IAA had different effects on the expressions of these genes.