Expression And Characteristics Of The Exogenous CpTI Gene In Transgenic Lines Of Apples

Apple is one of the most important fruit trees in the world. The research on genetransformation in apple has been conducting since 1989. Then some of the foreign geneswere transferred into apple rootstocks and cultivars successfully. But the studies weremainly focused on the process of the gene transformation, and few studies on expression,characteristics and inheritance of the exogenous genes were found. The study in theaspects of these issues would benefit to the application of gene transformation in appleand others.The transgenic lines of apple (Malus domestica Borkh.) carrying exogenousCowpea Trypsin Inhibitor (CpTI) gene including Royal Gala, Orin, Jonagold and Fujiwere used to study the expression of the CpTI gene in DNA, RNA and protein levels byusing the techniques of PCR, RT-PCR, FISH, electrophoresis and feeding the Helicoverpaarmigera larvae with the leaves. Meanwhile, the characteristics of the pollen and fruitsfrom the transgenic lines were evaluated. The inheritance of the CpTI gene was studiedby the reciprocal cross. The conducting effect of the report gene (Neomycinphosphotransferaseâ…¡, Nptâ…¡) between the rootstocks and scions was studied by usingmicro-grafting. The main results were as follows:1. The presence of exogenous CpTI and nptâ…¡genes in transgenic lines conservedfor 6～8 years by subculture at normal temperature were studied by the techniques ofPCR analysis and Kanamycin resistance evaluation. The PCR analysis results showedthat all transgenic lines could generate the expectable band. The leaves of two linespresented yellow when the plantlets cultured on the medium with 50 mg/L Kanamycin,which indicated the lack of the Kanamycin resistance, and the others could grownormally.2. The preliminary FISH technique system on apple was set up. The signals wereobserved successfully on the cell nucleus of the transgenic lines by using the probe of CpTI gene.3. The feeding experiment showed that some transgenic lines had the effect onrestricting the larvae growth or killing the larvae. There were two lines had the strongestaffection by killing the larvae of 100%. Twenty eight lines showed positive value of themodified death rate. And the modified death rate of the eleven lines was higher than orequal to 50%. It indicated that those lines had the ability to restrict the growth of the larvaeor kill the larvae. The weight of the larvae that fed by the leave of four lines showedsignificant difference from the control.4. The activity of the midgut chymotrypsin-like enzyme of the larvae after fed by theleave varied from the transgenic lines. The activity was significant difference from thecontrol when the larvae were fed by the leave collected from nine lines. That indicated thoselines had certain capacity of restraining the growth and development of the insect.5. Testing of the expression of exogenous CpTI by RT-PCR analysis in sixty linescultured in vitro indicated that there were high expression in forty three lines, and theexpectable band in seventeen samples was dim. The CpTI gene fragments fromtransgenic line Gala 32 was amplified, recoveryed, cloned and sequenced. The BLASTresult of the nucleotide acid sequence showed that the obtained gene fragment was highlyhomologous with phaseolus vulgaris trypsin proteinase inhibitor gene and tieganqingtrypsin inhibitor (TI) gene, and the degree ranged was 100%. The protein analysisshowed that the obtained gene fragment was highly homologous with proteinaseinhibitor-cowpea.6. The optimal protocol of Capillary Zone Electrophoresis (CZE) analysis for appleleaves was that: extracting the protein form leave by using improved ace-sediment method;setting the voltage as 20kV and the temperature as 25℃; setting the isoelectric focusingtime as 17 minutes and the wavelength as 280 nm. The result of CZE showed that at the7.65 minutes the exceptional protein brands were founded in the four transgenic lines ofsixty.7. The in vitro pollen germination rate for transgenic apple was 24.47% at 12 hours,lower than that of the control (62.05%). But the exogenous CpTI gene could increase theKanamycin resistance of pollen. The shape, size and ornamentation of pollen grains observedby SEM showed no difference between the transgenic apple and non-transgenic controlapple.8. Neomycin phosphotransferase (nptâ…¡) activities were detected in the most transgenicapple fruits, but the activities varied from the fruits with strong in 71.43%, weak in 21.43%,and no activity in 7.14%. The result of CZE for the fruits showed that the types of protein in transgenic Gala were few than those in non-transgenic control Gala.9. The embryos collected from the fruits by crossing between transgenic andnon-transgenic Gala and Fuji were cultured in vitro for new plantlet's generation. Thenthe Kanamycin resistance ability of the plantlets was evaluated. The inheritance patternsof F₁ plants showed about 1:1 segregation ratio, in accordance with Mendelian rule ofdominant gene with singal locus.10. For micro-grafting, the higher survival grafting rate was obtained when the plantletsthat subcultured for 30 days with vigorous adventitious shoots and the scion with 2～4leaves were used. The moderate increasing of the concentration of BA in the medium couldincrease the survival grafting rate. The suitable medium for micro-grafting in vitro wasMS+BA1.0mg/L+NAA0.05mg/L with a highest grafting rate of 83.3%. Meanwhile, noeffect was found on the survival grafting rate due to the exogenous gene transformation inapple. The exogenous nptâ…¡gene was only expressed in the transgenic explants and noconductivity was found between the scions and rootstocks by micro-grafting.