Molecular Detection And Traits Test Of Transgenic Ramie(Boehmeria Nivea L.Gaud) With P_SAG12-IPT Gene And B_t Gene

Ramie(Boehmeria nivea L.Gaud),one of the traditional fiber crops,had plaid an important role in our agriculture production.With development of society and market demand,ramie production faces a challenge,such as increment of population,decrement of farmland year by year.Thus,it would be a effective way to enhance per unit yield for the continued advance of ramie production.For improving ramie yield and quality by traditional breeding difficultly at present,plant genetic engineering provide a new method for ramie breeding,it is hopeful to obtain novel cultivars by introducing goal gene into ramie plants.P_SAG12-IPT(IPT gene with the SAG12 promoter) gene could autoregulate cytokinin production and delay leaf senescence.Bt(Bacillus thuringiensis) insecticidal gene encoded Bt toxin protein was applied on the most large scale in agriculture now.The two genes were introduced into ramie strain "5041" and cultivar "Luzhuqing" plants respectively,and an array of transgenic lines were obtained after we established ramie transformation system successfully.This paper carried out molecular detection on transgenic plants with P_SAG12-IPT and Bt gene,the results confirmed that the two gene had been integrated into ramie genome and could transcript normally.Photosynthetic characteristics of T₀ transgenic lines with P_SAG12-IPT gene were measured,the insect-resistance of the T₀ transgenic lines with Bt gene was bioassaied in the lab. Agronomic characters and quality character of all T₀,T₁ transgenic lines were tested.The main results in this study are as follows:1.T₀,T₁ transgenic lines were detected by PCR and Southern blotting analysis,and each line had got positive plants.The PCR positive ratio of Nptâ…¡gene was 80.1%,the PCR positive ratio of P_SAG12-IPT gene was 69.8%and co-transformation ratio was 66.1% among T₀ transgenic ramie strain"5041" plants,whereas the PCR positive ratio of Nptâ…¡gene was 73.8%,the PCR positive ratio of Bt gene was 33.4%and co-transformation ratio was 33.4%among T₀ transgenic cultivar "Luzhuqing" plants.The behavior of exogenous genes in both strain and cultivar were different.Mendelian inheritance of PCR detection was showed single dominant locus and proved by Southern blotting analysis in T₁ of all transgenic plants.Finally,we got T₀,T₁ transgenic plants,which would provide material basis for further test of physiology and characters.2.Light compensation point(LCP),apparent quantum yield(AQY) and dark respiration rate(Rd) of T₀ transgenic plants with P_SAG12-IPT gene were tested in the field in 2007,and they showed significance difference by compared with non-transgenic control.Part of the transgenic lines showed a single-peak curve,and the others showed a double-peak curve,but control always showed a single-peak curve in the photosynthetic rate(Pn) diurnal changes,the content of Chlorophyll(Chl) b of the control was higher than that of the transgenic lines,but the Chl a/b of the transgenic lines were higher than that of control in the first season and the second season in the fiber development period. Correlations between Pn and the major physiologic factors were analyzed and the results showed that different correlation coefficient existed in the first season,the second season and the third season.In addition,the endogenous cytokinins content of T₀ P513 and P504 line were significantly higher than that of control,but the major morphological traits were abnormal,there were no significant variation between the other lines(T₀ and T₁) and control.The integration of P_SAG12-IPT gene resulted in the changes of physiological traits.3.Bt transgenic lines inhibited obviously the herbst larva growth in the lab.All transgenic lines exhibited insecticidal activity,the mortality of larva fed with SBL2 line reached 38.7%,the mortality of pupae fed with SBL3 line reached 24.8%,the misshapen rate of the adult fed with SBL2 line reached 40.0%.The herbst could distinguish non-Bt ramie leaves from Bt ramie leaves when two kinds of leaves were fed synchronously. Moreover,the herbst prefers to feed on Bt ramie leaves.T₁ Bt transgenic lines and control were planted in the field where herbst took place seriously average year,the results showed that non-Bt ramie leaves were fed on by larva,even very few had been eaten up, but Bt ramie grew normally.4.The morphological traits of P_SAG12-IPT lines showed variation at some extent by compared with control,for example,there were many branches in middle section of P504 stem;the edge of the whole P513 leaves rolled up from bottom to top in late development period,part of lines exhibited inbred sterility.Agronomic characters and quality character of P_SAG12-IPT lines showed significantly variation:major character targets among P502 line were better than that of the cultivar 5041,Part of character targets were better than that of CK but any character targets were less than that of CK among major lines,There were even specific variance among the very few lines.T₀ Bt transgenic lines maintained the main traits compared to non-Bt ramie,two exogenous gene did not affect ramie heredity.5.The favorable transgenic lines were obtained in this study.P502 line was significantly superior to the control,such as plant height,rate of effective tiller,raw fiber weight,fiber fineness,it would be a excellent breeding material,raw fiber weight of P506 line and P512 line,fiber fineness of P513 line and insect resistance of SBL2 line were better than that of control respectively,these lines would become breeding middle materials.