Study On The Antisense Vector Of Rarl Gene Genetic Transformation Of Chewing Cane

The world's major chewing cane producing countries generally believe that disease is one ofthe biggest threaten to the production of chewing cane. Since traditional breeding technologiescan't obtain substantive progress on chewing cane diseases resistance, the development oftransgenic technology provides technical support as well as pointed out the path of developmentfor the breeding of chewing cane. In recent years, the rise of broad-spectrum diseases resistancegenes has brought about a new way for plant breeding of disease resistance, it especially candirectly induce or enhance the plant's own defense mechanisms, which have properties of strongfeasibility and no species specificity, and can achieve the effect of "once and for all".In this study, the antisense vector of Rar1gene had been constructed with the maize Ubipromoter, Agrobacterium tumefaciens strain LBA4404mediated Rar1antisense genetransformed Ningde, Fuan chewing cane and study on the fuction of Rar1gene. The HptIImarker gene had been detected in transgenic plants, the Hyg allergy test of the leaves and theGUS staining had been done with the15transgenic plants. Finally, the transcriptional level anddisease resistance physiological and bio-chemical indexes were tested; results indicated that theimport of antisense Rar1gene played a part in the disease resistance of fruit sugarcane. Themain results of this study are as follows:1. Chewing cane Rar1gene primer was designed and PCR amplified a size of approximately300bp of the Rar1gene cDNA fragment. Reversely connected the fragment to the intermediatevector PupTn in the downstream of Ubi promoter, restriction enzyme HindIII and EcoRV doubledigest the vector, the fragment was connected into the expression vector pCAMBIA1301, andthen transformed into Agrobacterium tumefaciens strain LBA4404, strain of transformation wasnamed LBA4404-anti-Rar1.2. Apply the agrobacterium-mediated transformation method to transform chewing cane,19positive plants had been detected within the173transformation seedlings by PCR analysis of theHptII marker gene.15transgenic plants had been determined by GUS staining,14of the15plants is Ningde chewing cane,1is Fuan.3. Transgenic chewing cane plants inoculated with Colletotrichum falcatumwent, it showedthat resistance of Colletotrichum falcatumwent were declined in the transgenic plants.4. Semi-quantitative RT-PCR detection of part of transgenic plants, results showed that: thetranscriptional levels of antisense Rar1transgenic plants of the Rar1gene have different degreesof reduction, down to63.9%of the control in Average; After inoculating Colletotrichumfalcatumwent, transcription level of chewing cane have increased in contrast with previous, it down to82.2%.5. After inoculating Colletotrichum falcatumwent in the transgenic plants, severaldisease-resistant relevant physiological and biochemical indexes have been detected,the resultsshowed that: the resistance related enzyme activity of SOD and POD was inhibited and downregulated, while no significant impact on the resistance relevant substance of MDA. Thisdemonstrated that the chewing cane Rar1gene has played a part in the pathway of reactiveoxygen resistance reactions.