Cloning And Characterization For Rar1Diseasere Sistance Gene In Sweet Potato

As we know, sweet potato is not only an essential kind of food in daily life, but also plays an significant role in both agriculture and industry. However, insect pest has largely influenced the quantity and high quality of sweet potato. It is demonstrated that broad-spectrum disease resistance attained by using SAR related gene, such as NPR1, is an effective way against pathogen attack in previous research. Here, we would achieve full-length cDNA of one of the key regulators in SAR response Rar1from sweet potato by making use of RT-PCR and RACE by Jinshan57as materials. At the same time, over-expressed vector would be constructed with this isolated gene,and it was transferred into model plant tobacoo by means of Agrobacterium,which may provide new theory evidence for its mechanism of disease resistance of this kind gene in sweet potato. The main experimental results are as follows:1. Separation and clone of Rar1:In this work,the cDNA full-length of Rar1was obtained by using RACE and RT-PCR,which length is672bp and contains222amino acid.It contains CHORD-Ⅰ,CHORD-Ⅱ,which nucleotide sequence is CQRIG CNAFFTEDDN PEGSCTYHDS GPLFHDGMKE WSCCKKRSHD FSLFLEIPGC KTGKH and TCKNK GCGKTFTEKE NHDTACSYHP GPAIFHDRMR GWKCCDIHVK EFDEFMAIPP CTKGW,and CCCH domain.What's more,it has the most homology of the nucleotide sequence with tobacco,to99%.2. The mechanism of Rarl expressing in sweet potato:It is confirmed that Rarl can express in different tissue by RT-PCR in different levels,such as root,stem,leaf,T which belong to constitutive expression.The research investigates the transcription level of Rar1show that the expression of Rar1can be improved after inducement by SA in appropriate concentration in different time by RT-PCR.3. The translation of Rar1gene into tobacoo:over-expressed vector is constructed by using pEGC5941as original vector.And it's transferred into tobacoo by Agrobacterium.There are20transgenic tobacoo seedings,which are real transgenic plant among them,and the probability of translation reaches50%.We found that the expressions in transgentic plant reach9times than blank plant by Real-time PCR.