Transformation and functional verification of Cry5Aa in cotton

Most of the cotton bollworm-resistant genes applied in cotton are more than 20 years and they all belong to Cry1Ab/c family, but the insect-resistant effects of Cry5Aa on cotton were rarely reported. The possible risk of resistance is increasing. The study synthesized a novel bollworm-resistant gene Cry5Aa artificially based on preferences of cotton codon. The new gene was transferred to cotton through the method of pollen tube pathway. The transgenic strains were identified by kanamycin test in field and laboratory PCR analysis. Meanwhile, an insect resistance test was conducted by artificial bollworm feeding with transgenic leaves and GK19 was used as a control in this study. Results showed that rate of positive transgenic strains with kanamycin resistance in the first generation (T1), the second generation (T2) and the third generation (T3) respectively were 7.76%, 73.1% and 95.5%. However, PCR analysis showed that the positive strain rate in T1, T2 and T3 were 2.35%, 55.8% and 94.5%, respectively. The resistant assay of cotton bollworm showed that the mortality rate of the second, third and fourth instar larva feed by the transgenic cotton leaves, were 85.42%, 73.35% and 62.79%, respectively. There was a significant difference between transgenic plant of Cry5Aa and GK19 in insect resistance. Finally, we also conducted the further analysis of gene expression patterns, gene flow and the effect on non-target pest in the study. The results showed that Cry5Aa gene had less environmental impact, and Cry5Aa has been transferred successfully and expressed stably in cotton. Therefore, the novel bollworm resistance gene can partially replace the current insect-resistance gene of Lepidoptera insects.

Bacillus thuringiensis (Bt) transgenic insect-resistant cotton has been widely planted in China, and this has resulted in the efficient control of cotton bollworm populations with reduced use of insecticides 1,2 . However, evolution of resistance by bollworm(s) threatens the continued planting of single gene Bt cotton 3,4 . Co-expressing of different types of insect-resistant Bt genes in cotton could alleviate the resistance of bollworm to transgenic insect-resistant cotton and extend the lifespan of transgenic cotton.
Cry5Aa is a member of Bt family, is a crystal protein produced by the Bacillus serotype 5 , highly toxic to rootknot nematodes and some lepidopteran pests. Lots of studies have shown that Cry5Aa shares a three dimensional structure with Cry1Aa, but it has long insertions in a2 of domain I. The particular extension may involve in pore formation and specific determination, and some of the rings in domains II and III are exposed, which makes it important for the design of high-efficiency bio-nematicides 5 . Transferred Cry5Aa into maize and potato could or has improved plants resistance to nematodes. The binding site of Cry5Aa to the nematode glycolipid receptor oligosaccharide fragment or fragments is between crystal protein domain I and domain II, because the interaction between Cry5Aa and oligosaccharide fragments is mainly conceived, followed by hydrogen bond energy, and static electricity. The action energy is zero, and a stable complex can be formed between them, which are more favorable for understanding the toxicological mechanism of the crystal protein Cry5Aa of B. thuringiensis and improving the nematicidal activity of Cry5Aa 6 .
In view of the fact that Bt gene has a good toxicity effect on lepidopteran pest, and the fact that Cry5Aa has not been studied in cotton, the purpose of our study was to transform the recombinant plasmid which contain the novel Bt insect-resistant gene, Cry5Aa, into the cotton by using the pollen tube pathway method.
We screened and identified the progeny using molecular biology method to verify the transfer of Cry5Aa Bt gene. We also conducted the further analyze of its expression patterns in cotton and analyze gene flow in environment and the possible effect on non-target pest. This study provided a new exploration for cotton transgenic insect resistance and lays the foundation for the creation of new insect-resistant germplasm resources. Pollen tube pathway. The methods of pollen tube pathway were defined as described by Zhao et al. 7 . The plasmid DNA was extracted, purified and diluted to the concentration of 100 μg/μL. On the next day of flowering, the flowers of the first and second fruit nodes of each fruit branch were selected as the objects of transgenic operation. The petals were stripped off and the plasmid was pushed into fertilized ovary with a 50 μL medical micro injector and each ovary was injected with 5 μL of diluted plasmid.

Kanamycin test.
After harvesting the seeds of T 0 generation, we sowed them in Sanya, Hainan Province, China in November 2011. We applied floating nursing for seeding and transplanted them to the field when they reached the three-leaves stage. We employed kanamycin to identify transgenic Bt cotton plant in the field experiment because the selection marker of NPT II involved in the plasmid produces kanamycin resistance in the leaves of transgenic cotton plants. The expanded leaves at seedling stage were scribbled with a writing-brush soaked in 1.6 μg/μL of kanamycin solution. Result was revealed three days later because all of leaves of nontransgenic plants showed apparent yellow plaque whereas the leaves of Bt transgenic plants remained green without any symptom.
Genomic DNA extraction and PCR analysis of the transgenic plants. The total DNA of cotton leaves were extracted by the method of CTAB. Based on the Cry5Aa gene, the primers were designed by Primer 5.0, just as follow: 5′-GCC ATC CAG GTT GCT ATC T-3′, 5′-GCT GAC GAA AGA ATG CTG A-3′. The target gene was 697 bp long. The total volume of the PCR reaction system was 20 μL. The concentration of template DNA was 10 μg/μL. The content of template DNA in reaction system was 20 μg. In this reaction, we added 2 μL of 10× taq buffer and a touchdown protocol was employed with the annealing temperature about 95 °C, following by 30 cycles at 58 °C and a 45 s of extension time at 72 °C for all cycles. After that, samples were conserved at 4 °C.
The analysis of Bt toxic protein in different organs and tissues. According to specific peptide sequence CNPNQPCKDDLDRV, the specific antibody was prepared by Nanjing Kingsri Bioengineering Co, Ltd. The total protein was extracted from cotton leaves at different stage by TCA-acetone precipitation. A standard curve was produced with 0.25-4.0 × 10 -6 μg/μL of Cry5Aa crystal protein and PBST buffer was used as a control. OD value of Bt toxic protein extracted from different organs and tissues of transgenic cotton lines were detected by ELISA assay. The contents of toxic proteins in different cotton organs and tissues were obtained according to the standard curve.
The test of insect-resistance. The cotton bollworm resistant assay of four Cry5Aa transgenic line including JX0010, JX0020, JX011 and JX012 was conducted. Jin 1 and GK19 were used as negative and positive control, respectively. Jin1 is the receptor and GK19 is a Cry1Ac Bt transgenic cotton variety widely planted in China. At the peak of the second, third and fourth generation of bollworm larvae, 20 cotton plants were randomly selected from each transgenic line and the control, and the leaves on the top of each plant were collected, marked, and brought back to the room in an ice box. The collected cotton leaves were put into a plastic box with a diameter of 6 cm and a height of 4 cm. One leaf was placed in each box. Five 1-day-old cotton bollworm larvae were injected These seeds were planted in the greenhouse of Hunan Agricultural University in Changsha, Hunan. After that, the genomic DNA of leaves at seedling stage were extracted and identified by PCR with Cry5Aa Bt specific primers and rate of positive Cry5Aa seeds to all seeds were calculated. Finally, gene flow of Cry5Aa was determined according to spreading distance of its pollens.
Effect of Cry5Aa on non-target pests. The experiment was conducted in 2010 at the experimental station of Hunan Agricultural University, Changsha, Hunan Province. Four non-target pests except the target pest bollworm including cotton aphid, miridae, two-spotted spider mite and cotton thrip were investigated every 20 days from May 20 to September 20 in field planted with Cry5Aa transgenic line JX0010 and non-transgenic control Xiangmian10. The Cry5Aa transgenic line and the control were both cultivated and managed in accordance with cotton cultivation technical standard of Hunan Province.

Results
Transformation of Cry5Aa to cotton. The plasmid containing the Cry5Aa gene was injected into young cotton bolls by pollen tube pathway and 0.6 μg/μL of gibberellins solution was sprayed onto bract for boll protection. The average setting rate of bolls reached 73.3%. A total of 2529 seeds were obtained for further identification (Fig. 1).

Kanamycin test and PCR analysis of transgenic lines.
After the seeds of To generation were harvested in 2010, they were planted at the research station of Hunan Agricultural University in Sanya, Hainan during the winter of that year. Total 1829 seeds of T 1 generation germinated normally while the other seeds failed to survive. The expanded leaves were sprayed with 1.6 μg/μL of kanamycin solution at seedling stage for preliminary identification. Total 142 plants without yellow plaque indicated that the positive rate of Cry5Aa transgenic line by kanamycin test was 7.76%. Meanwhile, PCR analysis was performed after genomic DNA extraction from transgenic plants. Specific band were identified in 140 plants showed that the positive rate was 7.4% by PCR analysis (Fig. 2). There was no significant difference between kanamycin test and PCR analysis proved that kanamycin test could be a simple, cheap and efficient technique for gene identification at least for cotton.
The seeds of T 2 generation were harvested from positive T 1 transgenic plants. A total of 845 T 2 plants were obtained at cotton base of Hunan Agricultural University in Changsha, Hunan in the same year. There were 618 positive plants without yellow spots after spraying with kanamycin solution. While the positive rate of transgenic line was 73.1% by kanamycin test and the positive ratio detected by PCR was 73.45% (Fig. 3). The expression character of Cry5Aa. Since the Cry5Aa is a novel Bt gene, there was no any ideal detection method could be applied. A specific antibody to Cry5Aa toxin protein was synthesized for ELISA analysis. The expression level of toxic protein in various tissues from T3 generation of transgenic line JX0010 at different stages were determined by the method of ELISA.
A standard curve was produced with purified Cry5Aa crystal protein and it was diluted to the concentration of 0.25, 0.5, 1.0, 2.0 and 4.0 × 10 -6 μg/μL, respectively by PBST buffer. PBST buffer was used as a blank control. It can be seen that the regression equation of the standard curve for Cry5Aa toxin protein was Y = 0.4093x + 0.1496. The correlation co-efficient R2 was 0.998 which indicates that OD value was significantly correlated with the content of Cry5Aa standard protein.
Through the ELISA detection of Cry5Aa toxin protein, the protein expression level of various stages and different tissues were shown in Fig. 4. Cry5Aa protein content of the leaves at bud stage was 1131.59 × 10 -6 μg/μL and it was the highest expression level. However, toxin protein content of small bolls at boll stage was with the lowest level of 526.45 × 10 -6 μg/μL. The results showed that Cry5Aa toxin protein was with the highest expression level at the bud stage among all developmental stages which explained its time distribution characteristics. In terms of the spatial distribution character of the novel Bt protein, it displayed the highest expression level in leaves among all tissues.

Determination of bollworm-resistance of Cry5Aa transgenic lines.
The results showed that the adjusted death rate of fed cotton bollworm of four Cry5Aa transgenic lines including JX0010, JX011, JX012 and JX0020 was 82.00%, 100%, 69.50% and 76.50%, respectively (Table 1, Fig. 5). However, that of the control GK19 was only 68%. The adjusted death rate of fed cotton bollworm of transgenic line JX0010 and JX011 increased significantly at 0.05 level compared with the control and the damage degree index of the leaves was 1. There was no significant difference between the other two transgenic lines JX012, JX0020 and the control with the same damage degree index of 2.  Effect of Cry5Aa on non-targeted pests. The experimental results illustrated the populations of all four tested non-target pests including cotton aphid, miridae, two-spotted spider mite and cotton changed among different growth and development stages. However, there was no significant difference between Cry5Aa transgenic line JX0010 and the non-transgenic control Xiangmian10, which maybe indicated that the novel Bt gene had less influence on these non-target pests.
Cotton aphid. During the entire developmental stage of cotton, the number of cotton aphids changed significantly. The damage extent of cotton plants affected by cotton aphid in transgenic cotton was even more serious than the control, but the difference between them was not significant (Fig. 6).
Miridae. The population of miridae in transgenic species and non-transgenic cotton are shown in Fig. 7.
Miridae appeared in late June and reached the maximum population in late July. While the increasing trend of the transgenic line and the control was similar. Cry5Aa transgenic line exhibited lighter damage extent compared with the control, but there was no significant difference between them. 68.00 ± 2.00aA 0.00 2 Figure 5. The bollworm resistances of transgenic line. www.nature.com/scientificreports/   www.nature.com/scientificreports/ Two-spotted spider mite. The population of two-spotted spider mite in both transgenic and non-transgenic cotton showed similar changing trend during developmental stages (Fig. 8). The pest occurred in late June, then the population increased gradually and reached to the peak value in late July. Compared with the control, the damage caused by two-spotted spider mite in transgenic lines JX0010 were slighter, but there was no significant difference between them.
Cotton thrips. Cotton thrips occurred in late May, its population reached to the peak value in late July, and the population size decreased gradually (Fig. 9). There was almost no difference in the degree of damage between transgenic strain JX0010 and the control.

Discussion
In 1983, Guangyu Zhou first transferred the foreign DNA into cotton through the pollen tube pathway method to obtain transgenic cotton plants with resistance to blight 8 . Bt insect-resistant transgenic cotton were obtained by this method for the first time was reported in 1991 9 . The pollen tube pathway method has the advantages such as no genotype limitation, no need of tissue culture, easy operation for large scale of plant transformation, etc 10 . The transformation rate was as high as 7.4% in this study proved the reliability of the method. Of course, the pollen tube pathway method is time consuming and requires rich experience of fertilization in cotton. Moreover, the most appropriate time for transformation is 18-48 h after pollination 11 . In addition, the conclusion can be drawn by this study, that the delayed transformation process in the early August combined with gibberellin spraying onto bracts can significantly reduce the shedding of cotton bolls, and effectively increase the transformation rate.
The novel Bt gene of Cry5Aa hasn't been reported the introduction to cotton. The specific primers were designed for PCR that used to confirm the transformation of the exogenous Cry5Aa in transgenic cotton. Firstly, the transgenic lines were preliminarily identified by kanamycin test in field. Then those positive transgenic plants were confirmed by laboratory PCR analysis. The positive rates identified by the two different methods were approximated proved that kanamycin test could be an effective, convenient and fast methods for transgenic confirmation. Moreover, high concentrations of kanamycin used in this study, which may be necessary for transformation confirmation and can significantly improve the accuracy of the assay.
The results of ELISA assay confirmed that Cry5Aa was indeed expressed in various tissues at different developmental stages of transgenic line JX0010. There was a significant difference in the expression levels of various tissues, which is consistent with the previous studies on expression character of other Bt genes. This study confirmed that the insecticidal effect of Cry5Aa transgenic lines on cotton bollworm and their insect resistances had a tendency to decrease along with the growth and development process of cotton plants [12][13][14] .
The bollworm resistance test of four Cry5Aa transgenic lines showed higher insect resistance compared to the control. Among four Cry5Aa transgenic lines including JX0010, JX011, JX012 and JX0020, the two transgenic lines JX0010 and JX011 significantly increased the bollworm resistances and the damage degree index of the leaves was 1, whereas there was no significant difference between the other lines JX012, JX0020 and the control with the same damage degree index of 2.
Controlling the gene flow of GM crops can reduce the negative impact on non-transgenic crops 15 . The mechanism of action of Bt on target pests is that companion crystals are produced during the sporulation stage, and the crystals are taken by lepidopteran insects. After eating, it is activated by the hydrolysis of intestinal enzymes, and Howard toxin can bind to the midgut specific receptors to cause the formation of mesenteric pores and cause insects to die 16 .
Studies have shown that due to the incompatibility of hybridization, unoccupied flowering or geographical isolation, the possibility of gene drift or gene introgression of Bt gene transgenic cotton to wild cotton or relative plants of land cotton is basically non-existent 8 . It is important to determine the gene flow distance of the novel www.nature.com/scientificreports/ Bt gene by field experiment. The results that the maximum pollen spreading distant of Cry5Aa transgenic line was limited, indicates that the Cry5Aa has less impact on the environment. During the growth and development of Cry5Aa transgenic cotton, it was found that the populations of all four non-target pests such as cotton aphid, miridae, two-spotted spider mite and cotton changed along with various development stages of transgenic cotton plants. However, there was no significant difference between Cry5Aa transgenic line JX0010 and the non transgenic control. Previous paper reported that Cry5Aa toxin has a certain inhibitory effect on blind mites and double-spotted spider mites 17 . Some other factors such as cotton varieties, management measures, and geography also play vital roles in impact of transgenic lines on non-target pests In the future, prevention of possible harm on non-target insects caused by transgenic Bt cotton is as important as control of cotton bollworms.

Conclusion
In this study, the transformation and functional confirmation of Cry5Aa was introduced in detail. Cry5Aa, as a novel Bt insect-resistant gene, can significantly improve the resistance of transgenic plants to cotton bollworm, and can partially replace the current mainstream lepidopteran resistance gene. At present, the safety of transgenics is the most concerned issue. This study confirms that Cry5Aa can be safely applied to production and has controllable environmental impact. Due to the effective control of lepidopteran pests such as cotton bollworm and the economic and ecological benefits of the production, the transgenic Bt cotton has been accepted by more and more people, which has made it impossible to maintain the sustainable development of cotton in the world.