| Cotton is one of the most important economic crops in agricultural production in China.It can be used as an important raw material in the textile industry and an important source of oil crops.In the modern production of cotton,pests are an important issue affecting cotton yield and quality.China’s annual losses caused by pests are about 12%-15%of the total cotton output.With the continuous development of genetic engineering technology,breeders use genetic engineering technology and traditional breeding methods combined with Agrobacterium tumefaciens-mediated method to introduce exogenous Bt gene into recipient cotton plants,and obtain more than 80%Transgenic Bt cotton;Thus effectively controlling the cotton bollworm and other pests on cotton production hazards,greatly reducing the losses caused by pests.Since 1994,China successfully developed the Bt gene price insect-resistant cotton since,so far,transgenic insect cotton has accounted for more than 93%of total cotton acreage.As transgenic insect-resistant cotton is not harmful to humans,livestock and the ecological environment,it has brought great economic value,ecological benefits and social benefits.This paper studies the following aspects and obtains the following results:1.Insect Resistance Identification of Transgenic Insect-resistant Cotton(1)PCR identification.The four transgenic insect-resistant cotton varieties YL02-1,GK19,G-6 and 33B were identified by PCR using the specific primers for the detection of Bt gene in China and America.The results showed that the Bt gene was included in the four cultivars,and the cultivars YL02-1 and GK19 were 456bp,which was the domestic price Bt insect resistant gene.The cultivars G-6 and 33B Bt insect-resistant gene primer design of the characteristics of the original fragment length of 310bp,that is,foreign production price Bt insect-resistant genes.(2)Identification of Bt Toxin Protein Content.The contents of Bt toxic protein in four combinations of sea and land hybrids and parental embryos were determined by ELISA.The results showed that the Bt toxic protein content of the F1 generation was higher than that of the insect-resistant parent,and only one combination of Bt-toxic protein was close to the insect-resistant parent,and reached the high resistance level(high resistance>450 ng/g)It was found that Bt gene was expressed in F1 generation.Compared with domestic and foreign insect-resistant parents and Fi hybrids,the Bt protein content of Bt-induced protein was not inferior to that of foreign varieties.(3)Identification of Biological Insect Resistance.The insect resistance test of the four cultivars YL02-1,GK19,G-6 and 33B was carried out by using Sumian 9 as the control varieties,and the dynamic resistance information of different indexes were obtained.Resistance information.The results showed that the comprehensive resistance value K of the four cultivars was between 35 and 41,the average resistance value PK was between 3.00 and 3.5,the comprehensive resistance level was the same,and there was no statistically significant Differences show that domestic and foreign insect-resistant cotton varieties have no significant difference in biological resistance to insects.2.Inheritance of Bt Gene in Insect-resistant CottonThe transgenic cotton seed germplasm was used as the parent and the non-insect resistant island cotton seed coat system to arrange four combinations of land and sea:YL02-1 × Hai7124,GK19 × V-1,G-6 × Hai7124,33B × V-1.And then Fi generation self-cross to obtain four combinations of F2 generation groups.The Bt gene was amplified by PCR,and the Bt gene was amplified by PCR.The results showed that the F2 population of the four combinations was Bt gene and the Bt gene alone were fitted with 3:1,respectively,indicating that the four insect-resistant cotton are controlled by the single dominant gene,which can be chromosomal location.PCR was performed by PCR-specific amplification of the DNA molecules in the F2 population of the four combinations.After the gel electrophoresis,the images of the target fragment were observed,and the statistics showed that the four groups of F2 populations were all fitted with the theory of 3:1 Separation ratio,in line with Mendelian genetic law.Is a genetic inheritance controlled by a pair of dominant genes.3.Construction of Genetic Map of Bt Gene of Insect-resistant CottonA total of 234 pairs of core primers were used to detect the near-isogenic pool in 26 populations of cotton.The F2 population was identified by four insect-resistant parents.After detection found:(1)In the[YL02-1 × Hai7124]F2 population,38 pairs of SSR polymorphism primers were obtained.The results showed that the labeled NAU2579 was linked to the Bt gene in YL02-1,and the known marker NAU2579 was located on chromosome 20 of cotton.The chromosome was located on both sides of primer NAU2912 About 50cM of other primers.Then,the polymorphism was screened by the parents,and the genotype of F2 population was detected by polymorphic primers.Linked genetic analysis was performed by JoinMap 4.0 software.17 pairs of polymorphic primers were linked to Bt gene.The Bt gene in YL02-1 was located on the 20th chromosome of cotton.The molecular markers were Gh564 and NAU3813,and the genetic distances were 2.8cM and 12.2cM.(2)In the[GK19 × V-1]F2 population,22 pairs of SSR polymorphism primers were obtained.The results showed that the labeled NAU2579 was linked to the Bt gene in GK19,and the known marker NAU2579 was located on chromosome 20 of cotton.The synthesis was located within about 50cM on both sides of the chromosome on the labeled NAU2912 Of other primers.Then,the polymorphism of the primers was screened by the parents.The genotype of the F2 population was detected by the polymorphic primers.Linked genetic analysis was performed by JoinMap 4.0 software.The results showed that 16 pairs of polymorphic primers were linked to the Bt gene in GK19 The Construction of the genetic map,GK19 in the Bt gene located between the labeled NAU3907 and NAU2579,the genetic distances were 2.4cM and 1.5cM.It can be seen,GK19 contained in the Bt insect resistance gene is also located on the 20th chromosome on the cotton.(3)In the[G-6 × 7124]F2 population,32 pairs of SSR polymorphism primers were obtained.The results showed that the primers NAU2912 were linked to the Bt gene in G-6,and the known marker NAU2912 was located on the 26th chromosome of cotton,and the chromosomes on both sides of the synthetic marker NAU2912 were found.About 50cM of other primers.The polymorphism of the primers was then screened by the parents,and the F2 genotype was detected by the polymorphic primers.Linkage genetic analysis was performed by JoinMap 4.0 software.The results showed that 17 pairs of polymorphic primers were linked to Bt gene in 33B.The Bt gene in G-6 was located on the 26th chromosome of cotton.The molecular markers were cgr6471 and dc60240,and the genetic distances were 4.5cM and 3.3cM,respectively.(4)In the[33B × V-1]F2 population,37 pairs of SSR polymorphism markers were obtained.The genotype of NAU2912 was linked to Bt gene in 33B.The results showed that the polymorphic primers NAU2912 were linked to the Bt gene in 33B.And the known marker NAU2912 is located on the 26th chromosome of cotton and synthesizes other primers located within about 50cM on both sides of the chromosome on which the label NAU2912 is located.The polymorphism of the primers was then screened by the parents,and the F2 genotype was detected by the polymorphic primers.Linkage genetic analysis was performed by JoinMap 4.0 software.The results showed that 17 pairs of polymorphic primers were linked to Bt gene in 33B.The target genes were located between markers NAU460 and dc60240,with genetic distances of 3.6cM and 2.0cM,respectively.It can be seen from the figure,33B in the Bt insect resistance gene is also located on the 26th cotton chromosome. |
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