Analysis Of Bt And EPSPS Gene Related Agronomic, Biochemical And Proteomic Difference In Glandless Cotton And Genetic Effects For Biochemical And Economic Traits In Bt Hybrid Cotton

Insect and weeds are the two major factors affecting cotton production. It is one of cost-effective methods to breed insect-resistance and herbicide-tolerant cotton cultivars using transgenic methods. The development of glandless cotton makes upland cotton becoming a new economic crop with multi-utilization of fiber, oil, food and feed. However, glandless cotton is more susceptible to pests than normal cultivars due to the absence of gossypol and other terpenoid aldehydes. Therefore, breeding Bt transgenic glandless cotton is benifical and a cost effective approach for its commercial utilization. Transgenic Bt hybrid cotton is widely used with the feature of insect-resistance, high yield and good quality. Based on the change of cotton growth and development, researches are still limited concerning the use of biochemical parameters to assess exogenous genes effects on glandless cotton lines and screen the promising parents and crosses. The present work was carried out by comparing two pairs of near-isogenic lines (NILs) of Bt and non-Bt and EPSPS and non-EPSPS glandless upland cotton in mineral uptake, photosynthetic performance, active oxygen metabolism and protein expression. In addition, diallel crosses using conventional upland cotton lines with transgenic Bt upland cotton lines were conducted to analyze their difference in phenotypic and genetic contribution ratios of biochemical traits to lint yield and the heterosis of yield, yield components and fiber properties and to further screen the promising parents and hybrids among Bt hybrid crosses and their parents lines. The main results are as follows:1. Agronomic and biochemical traits and protein expression were compared using a pair of near-isogenic lines (NILs) of glandless upland cotton differing in only by the presence of cryIAc gene (non-Bt variety-Zhong5629vs its Bt-transgenic near-isoline). The results showed that Bt isoline had higher plant height and rate of effective bolls, and more internal bolls than non-Bt isoline, while being lower in yield and number of bolls. Physiological analyses indicated that Bt isoline had lower chlorophyll contents and transpiration rate relative to non-Bt isoline, but higher in Ch1a/b, F0and Fm, and no significant difference in net photosynthetic rate (Pn), intercellular CO2concentration and stomatal conductance, and Fv/Fm. Content of soluble protein in Bt isoline was significantly higher at boll setting stage (BSS) but lower at initial flowering stage (IFS) compared with non-Bt isoline. The concentrations of Ca, Mg, Cu, Zn, Mn, and Fe at IFS, P and Cu at BSS in Bt isoline were significantly higher than those in non-Bt isoline, but reversely P, K and B at IFS, K, S, Zn, and Fe at BSS, respectively. Malonaldehyde (MDA) content at BSS and superoxide dismutase (SOD, EC1.15.1.1) activity at IFS and BSS were lower in Bt-isoline.2-DE analysis of the two NILs detected20differentially expressed protein spots, with4and16being up-and down-regulated in Bt vs. non-Bt isoline, respectively. These proteins were attributed to protein metabolism, defense response, transcription, energy metabolism, and cell structure. Among them, a core-neofusion protein spot as a selectable marker and neomycin/kanamycin resistance was specific expressed in Bt isoline.2. Physiological traits and proteomic expression were compared between EPSPS-G6transgenic glyphosate-tolerant glandless upland cotton (GT) and its non-transgenic wild type (non-GT, cv. Zhong5629). GT showed higher net photosynthetic rate at BSS than non-GT, with higher stomatal conductance (gs), intercellular CO2concentration (Ci) and transpiration rate (Tr) at the beginning of boll open stage (BBOS), but lower in Ci at BSS. Contents of Ch1a, Ch1b and Ch1a+b in GT were uniformly significantly less than those in non-GT at BSS and BBOS. Insertion of EPSPS gene had altered uptake of mineral nutrients:GT demonstrated less concentrations in N, Mg and K, but higher in P, Ca, Fe, K, Cu, Mn and Zn compared with non-GT. Contents of soluble sugar at BSS and BBOS and soluble protein at PFS and BSS in GT were significantly higher than non-GT; but GT had lower soluble sugar content at PFS and soluble protein at squaring stage (SS). GT recorded significantly higher activities in superoxide dismutase and catalase, but lower in peroxidase and ascorbate peroxidase compared with non-GT. Proteomic alteration in leaves of GT vs. non-GT was analyzed using2-DE coupled with mass spectrometry. Eleven differentially expressed proteins were identified, of which7and4spots being up-and down-regulated, respectively. GT showed up-regulated expression of RuBisCO large subunit, CP4EPSPS, and ATP synthase, but down-regulated glutamate-1-semialdehyde-2,1-aminomutase and manganese-stabilising protein, respectively.3. To examine the genetic contributions of biochemical parameters to lint yield at both antioxidant enzymatic activity and nutrient levels and to determine the major indicator traits for indirect selection on lint yield in Bt hybrid cotton lines, multivariable conditional analysis was conducted using a additive-dominance model. Six conventional upland cotton lines were crossed with3transgenic Bt gene lines using diallel mating design method. Nine breeding lines and derived16F1crosses were grown at the farmland of Cixi cotton research institute in Zhejiang province, China, in2010. Biochemical parameters at different developmental stages were analyzed, agronomic and economic traits were investigated and fiber quality characters were surveyed. Chi a and Chi b at SS and IFS, C/N raton at IFS and BBOS could be used as indicative physiological traits for selecting F1crosses, while Chi a and Chi b at full flowering stage (FFS) and BBOS, C/N raton at FFS for parents lines. N content at IFS, FFS, and BBOS, P and K contents at four stages were mainly affected by dominant effects, which could be used to choose F1crosses. Similarity, Parent’s lines have high additive effects in Chi a content, in C/N ratio and N content at SS, IFS and FFS, in K at IFS, FFS and BBOS, their hybrids F1have more lint yield. Bolls per plant, lint percent, micronarie and fiber length were mainly affected by additive effects, which suggested they could be improved by choosing pure lines. Additive effects contribution of lint yield was mainly from lint percent, while dominant effects contribution of lint yield from boll weight. Having high contribution ratio of additive effects to fiber strength, micronarie and fiber length, lint percent in parents could be used as indicative traits for selecting fiber quality of F1crosses. In short, Parent1(31) is an acceptable non-Bt line and parent8(B7kang) is an excellent Bt transgenic cotton with insect-resistance. Among16F1crosses, there are8F1crosses (H18, H28, H29, H37, H48, H59, H67and H69) with positive and significant lint yield.4. Four conventional upland cotton lines with five transgenic line using NCII design, and their20F1crosses were grown at the same location above in2011. genetic effects analysis showed that12biochemical parameters at three stages were mainly affected by dominant effects. K content in F1crosses at three stages had significant mean and better parents heterosis. C/N ratio had significant additive contribution ratio to lint yield at three stages. Branches, bolls, boll weight and lint yield were mainly controlled by additive effects, which suggested they could be improving by choosing pure lines. Additive variance contribution ratio of bolls for parents was mainly from external, middle and lower bolls on the plants, while for F1crosses from external and middle bolls. Additive contribution ratio of lint yield was from bolls and lint percent, while dominant contribution ratio from diameter, plant height and boll weight. Lint percent to fiber strength, plant height and bolls to micronarie, diameter to fiber length had largest additive contribution, respectively. In short, Parent3(Ci96-21) is an acceptable non-Bt line and parent9(huanggang kang) is an excellent Bt transgenic cotton with insect-resistance. Among20F1crosses, there are6F1crosses (H39, H16, H35,H28, H47, and H45) with positive and significant lint yield.5. Reciprocal crosses between one conventional upland cotton line (Ci-ZJ6) and five transgenic Bt gene lines was made to produce10F1hybrid crosses and all lines planted in2011at the same location above. Genetic analysis showed that Chl a at IFS and BBOS, Chl b at FFS and BBOS, Chl a/b ratio at three stages, N at IFS and FFS, P at FFS, K at FFS and BBOS, C/N ratio at IFS and BBOS, anti-oxidant enzyme systems (SOD, POD, CAT, APX) and MDA, were mainly affected by additive effects. While Chl a at FFS, Chl b at IFS, N at BBOS, P at IFS and BBOS, K at IFS, C/N ratio at FFS, were mainly affected by dominant effects. All12biochemical parameters in F1crosses had significant mean parents heterosis at three stages, but no positive and significant better parents heterosis. Diameter, external bolls, middle bolls, boll weight, lint percent, fiber length, micronaire and fiber strength were mainly affected by additive effects, which suggested those traits could be improved by choosing pure lines. Bolls additive variance ratio for parents was mainly from top and external bolls on the plants, while for F1crosses from central, lower, and external bolls. Additive contribution ratio of lint yield was mainly from branches, diameter, up bolls and lint percent. Lint percent to fiber strength and micronaire had largest additive contribution. Having significant additive effects in lint percent, boll weight, and fiber length and fiber strength. Parent1(Ci-ZJ6) is an acceptable non-Bt line. Parent4(E29) with better performance in bolls, fiber length and positive lint yield, and parent5(Tai kang) with more branches, bolls and positive lint yield, are excellent Bt transgenic cotton with insect-resistance. Among10F1crosses, there are3F1crosses (H13, H15and H41) with positive and significant lint yield. Among reciprocal crosses, H15and H51in diameter and fiber length, H12and H21in plant height, internal bolls and lint percent, H13and H31in lint percent had mean parents heterosis, respectively, which suggested that there were significant differences between conventional parent P1and five transgenic Bt cotton lines. Moreover, the overall performance in the crosses with female parent P1was better than in the crosses with female parents transgenic Bt cotton lines, which confirmed the possible existance of cytoplasmic inheritance in transgenic Bt hybrid lines.