The Research On Construction Of Intron-containing Hairpin RNA For PRSV And Their Application In Transgenic Papaya

Papaya (Carica papaya L.) is an important fruit in tropical and subtropical regions, which is famous for containing rich nutrients and papaya protease. Many factors affect the development of papaya industry, and Papaya ringspot virus (PRSV), is a major disease. PRSV causes significant yield losses in papaya. These problems are much harder to solve rely on conventional breeding methods, and it must depend on the method of biological technology. In order to study the broad-spectrum antiviral mechanism in papaya and develop transgenic papaya with durable PRSV resistance in south China, we used hpRNA anti-virus technique and genetic transformed the four different ihpRNA expression vectors into papaya plants throught pollen tube channel transformation technology, to provided a basis of theory and practice for papaya biotechnology research in China.The main results were as follows:1Based on the conservative sequence of CP gene of PRSV-HN strain, we developed a novel method to construct intron-containing hairpin RNA (ihpRNA) rapidly and efficiently throught asymmetric Overlap Extension PCR systerm. This method is a simple, rapid and economic (without any expensive reagent) technique for ihpRNA structure constructing. It is a one-step PCR in one tube without any intermediate purification. Based on the conservative CP gene (coat protein) sequence from hainan PRSV (PRSV-HN), we successfully generated10ihpRNA structure which target on difference sites of PRSV-CP gene, for respectively:hpRNA-Sl (9351-9705bp), hpRNA-S2(9406-9705bp), hpRNA-S3(9535-9705bp), hpRNA-S4(9590-9705bp), hpRNA-S5(9655-9705bp), hpRNA-S6(9720-10073bp), hpRNA-S8(9940-10073bp), hpRNA-S9(9974-10073bp), hpRNA-S13(9590-10073bp), hpRNA-S14(9655-10073bp).2Based on two rounds of Gene Splicing by Overlap Extension PCR (OE-PCR) technology, we developed a method to obtain mosaic ihpRNA structure. Its advantage is that it can target at several genes or multiple parts of one gene. Based on CP gene and VPg gene of PRSV-HN, we built one mosaic ihpRNA structure, named as hpRNA-CV. It targets on the two difference site of PRSV, which is promising to obtain broad-spectrum resistance and enhance the resistance to PRSV in papaya.3Using the pBI121and pCambia3300as background, we obtained four plant expression vector: pCambia-hpRNA-S5, pCambia-hpRNA-S8, pCambia-hpRNA-S6and pCambia-hpRNA-CV, which based on the structure of hpRNA-S5, hpRNA-S8, hpRNA-S6and hpRNA-CV.4The plant expression vectors with the ihpRNA structure were transformed into papaya plants (Sui zhong hong) through the pollen tube channel technology. The plasmid DNA was used as material in pollen tube channel technology platform. Through herbicide selection、 PCR detection, we got24plants of transfermation papaya, they are5plants of pCambia-hpRNA-S5,8plants of pCambia-hpRNA-S8,6plants of pCambia-hpRNA-S6and5plants of pCambia-hpRNA-CV. RT-PCR test results showed that the interest genes were stable expressed in transgenic plants.5Throught resistance test of PRSV virus poison attack and ELISA test, we got10resistance transgenic papaya plants. In detail:there are2transgenic plants of pCambia-hpRNA-S5,4transgenic plants of pCambia-hpRNA-S8,2transgenic plants of pCambia-hpRNA-S6,2transgenic plants of pCambia-hpRNA-CV. The tests show that the cumulant of virus decreased significantly than the control plants. That is to say that the cumulant of virus and plant resistance to PRSV have a negative correlation.6Northern blot results revealed that PRSV-CP gene was silenced in ihpRNA transgenic papaya plants, which enable the transgenic plants have obvious resistance to PRSV. The signals of Northern blot hybridization increased following the disease-resistant’s weaken of transgenic plants. The RNA accumulation of PRSV-CP gene in PRSV-resistant plants is significantly lower than the sensitive plants. That is to say the RNA accumulation of PRSV-CP gene in the transgenic plant showed a negative correlation with virus resistance.