|
|
Identification of Target Traits and Genetic Stability of Transgenic Cotton GGK2
|
| LIANG ChengZhen, ZANG YouYi, MENG ZhiGang, WANG Yuan, MUBASHIR Abbas, HE HaiYan, ZHOU Qi, WEI YunXiao, ZHANG Rui, GUO SanDui |
|
Scientia Agricultura Sinica
2023,56(17
):3251
-3260. DOI:10.3864/j.issn.0578-1752.2023.17.002
|
Abstract
（356）
HTML
（40）
PDF（pc）
（2506KB）（295）
Save
【Objective】The objective of this study is to confirm the target traits and genetic stability of transgenic glyphosate- resistant cotton GGK2 and provide technical support for its commercialization. 【Method】T3, T4, and T5 transgenic cotton plants GGK2 were subjected to insertion site-specific PCR, Southern blot, ELISA, bioassays in the laboratory and field, analysis of target herbicide tolerance, and investigation of nutritional constituents. 【Result】The results indicated that the target genes, GR79 EPSPS and GAT, were integrated into the cotton genome as single copies and stably inherited in GGK2 plants. In GGK2 cotton, GR79 EPSPS, GAT, and NPTⅡ proteins were expressed at different stages and in different tissues, with relatively high expression levels in the leaves. At the four-leaf stage, bud stage and boll opening stag, the expression levels in leaves were 128.7-192.4 µg·g-1, 24.4-35.0 µg·g-1, and 17.0-23.9 µg·g-1 fresh weight for GR79 EPSPS, GAT, and NPTⅡ, respectively. In the field, transgenic cotton GGK2 tolerated up to four times the recommended medium dose of glyphosate application. No significant differences were observed in agronomic traits and nutritional constituents compared to the control, Coker312. 【Conclusion】These data demonstrate that transgenic cotton GGK2 is genetically stable and highly resistant to herbicides. Therefore, it can be utilized for breeding high-glyphosate- resistant commercial cotton varieties.
|
|
Table and Figures丨
Reference丨
Related Articles丨
Metrics
|
|
|
The Creation and Characteristics of Cotton Germplasm Lines Transgenic 1174AALdico-2+CTP Gene with Excellent Glyphosate Tolerance
|
| WANG WanRu, CAO YueFen, SHENG Kuang, CHEN JinHong, ZHAO TianLun, ZHU ShuiJin |
|
Scientia Agricultura Sinica
2023,56(17
):3261
-3276. DOI:10.3864/j.issn.0578-1752.2023.17.003
|
|
Abstract
（267）
HTML
（10）
PDF（pc）
（2614KB）（820）
Save
【Objective】 Weeds are one of the factors limiting cotton growth in cotton production, which not only competes with cotton for nutrition, water, and light, affecting the growth and development of cotton but also the yield and quality of cotton. The aim of this study is to develop excellent cotton germplasms with high glyphosate tolerance by genetic engineering and provide technical support for the commercialization of glyphosate-tolerant cotton to realize chemical weed control and enhance the economic benefits of cotton production. 【Method】 To construct the cotton transformation vector, the EPSPS gene 1174AALdico-2 from Deinococcus radiodurans was connected to the chloroplast transit peptide (CTP), 35S was used as the promoter, and the two target genes were linked in series. The target gene was transformed into the CCRI 49 by Agrobacterium tumefaciens in vivo transformation, and the positive transformants were obtained. The positive transgenic plants obtained were evaluated for glyphosate tolerance, molecular characterization, agronomic and economic traits, in order to obtain outstanding transgenic cotton germplasms with excellent glyphosate tolerance, using the recipient cultivar, CCRI 49, and the non-transgenic NON isolated from the transformants during selfing as the controls. 【Result】 138 positive transformants were obtained by Agrobacterium tumefaciens in vivo transformation. All the transformants were tested for target gene PCR, Southern blot, Western blot, and ELISA detection etc., and 17 positive transformants with clear molecular characteristics and high expression of foreign genes were identified. Southern blot and nucleotide sequencing results revealed that there was great variation in insertion site and copy numbers among the 17 transformants. Among them, ZD131, ZD185, and ZD207 had single-copy insertion sites, and the foreign genes were located on D7, D13, and A12, respectively. The results of glyphosate tolerance identification revealed that the glyphosate tolerance of the three transformants, ZD131, ZD185, and ZD207, was stably inherited across three generations, and the glyphosate tolerance of the transformants was strong, with the ability to tolerate four times the recommended dose of glyphosate in the field. The agronomic and economic features assessment findings revealed that the ZD131, ZD185, and ZD207 grew properly and had excellent agronomic traits such as large boll, high lint percentage, and high boll setting, as well as their lint yield was higher than that of the receptor cultivar control and their fiber quality reached to the high-quality cotton level. 【Conclusion】 The EPSPS gene 1174AALdico-2 from Deinococcus radiodurans connected with the chloroplast transit peptide, and the two gene tandem together was transferred into CCRI 49 by Agrobacterium tumefaciens in vivo transformation technology. After a series of screens, three outstanding transgenic glyphosate-tolerant germplasms, ZD131, ZD185, and ZD207, were obtained. This method not only improves cotton's glyphosate tolerance but also improves the agronomic and economic aspects of transgenic materials.
|
|
Table and Figures丨
Reference丨
Related Articles丨
Metrics
|
|
|
Identification of Molecular Characterizations for Transgenic Cotton R1-3 Line of Glyphosate Tolerance
|
| MA YanBin, LI HuanLi, WEN Jin, ZHOU XianTing, QIN Xin, WANG Xia, WANG XinSheng, LI YanE |
|
Scientia Agricultura Sinica
2023,56(17
):3277
-3284. DOI:10.3864/j.issn.0578-1752.2023.17.004
|
|
Abstract
（245）
HTML
（11）
PDF（pc）
（3187KB）（215）
Save
【Objective】 To obtain the transgenic cotton by Agrobacterium-mediated method was the purpose using the new gene G10aroA with high glyphosate tolerance in our laboratory. Meanwhile, it was necessary to provide molecular characteristics of genomic integration of the exogenous gene for breeding utilization in future. 【Method】 The Agrobacterium-mediated method was used for transgenic cotton plants obtained via tissue culture with glyphosate herbicide. Western blot was utilized to detect the expression of exogenous proteins in different organs of transgenic cotton R1-3. The number of loci in cotton genomes were evaluated by Southern blot for detecting integration of the exogenous sequence from the pCAMBIA1300 construct. The flanking sequence near the insertion site was amplified by TAIL-PCR, which the extractions of DNA were cloned and sequenced. The location of the chromosome for the flanking sequences were compared and analyzed on the website of NCBI blast. 【Result】 Regenerated R1-3 cotton plants were successfully obtained by tissue culture depending on glyphosate screening. the specific protein coded by exogenous G10aroA gene could be detected normally via Western blotting in the leaves, bracts, flowers and stems separately, and the size of the exogenous protein around 46 kDa were also observed in this experiment. In addition, the result of Southern-blot confirmed that the exogenous fragment containing G10aroA sequences was single integration in the genome of transgenic cotton R1-3, in which the bands digested severally by KpnⅠ and EcoRⅠ endonucleases were distinctly observed near the position of 6 557 bp and 4 316 bp strips on the nylon membrane respectively. The analysis of flanking sequence alignment for the integration site was predicted to be located on the 11th chromosome of either cotton A or D genome, and the left and right boundaries of the insertion site were further located between 47 525 303 and 47 525 449 of the chromosomes. In addition, the specific identification for the fusion site showed that the target band of approximately 300 bp for testing left border junction, and a specific target band can be amplified about 600 bp for testing the right border fusion site. 【Conclusion】 In this study, we obtained R1-3 transgenic cotton plants that also have exhibited stable genetic characteristics of glyphosate resistance during the process of self-crossing breeding. The protein coded by G10aroA gene was about the size of 46 kDa that could be detected in different tissues of transgenic cotton R1-3 plant. Furthermore, the exogenous fragment including G10aroA gene was identified with a single location by southern blot in the cotton genomes, and the integration site was located at the 11th chromosome. The results of comparative analysis were predicted that a nucleotide sequence about 146 bp length was deleted at the integration of the genome.
|
|
Table and Figures丨
Reference丨
Related Articles丨
Metrics
|
|
|
Opportunities and Challenges for Developing Herbicide-Resistance Crops in the Post-Genomic Era
|
| WU YuanLong, HUI FengJiao, PAN ZhenYuan, YOU ChunYuan, LIN HaiRong, LI ZhiBo, JIN ShuangXia, NIE XinHui |
|
Scientia Agricultura Sinica
2023,56(17
):3285
-3301. DOI:10.3864/j.issn.0578-1752.2023.17.005
|
|
Abstract
（365）
HTML
（22）
PDF（pc）
（1743KB）（232）
Save
Global agriculture is facing severe challenges, and breeding technology is the foundation and key to the development of the seed industry. Gene editing technology refers to the precise modification of target genes to achieve deletion, insertion, and replacement of specific target gene fragments. It can precisely modify target genes or introduce certain excellent genes into crops to produce crops with excellent agronomic traits, which has great potential in molecular design breeding and is of great significance to ensuring food security. Weed damage has a huge impact on the yield and quality of crops. To control weed damage efficiently, safely and sustainably has always been a hot research topic. Currently, more than 200 types of chemical herbicides have emerged in the global market. Using chemical methods to control weeds has become an important part of modern agriculture, and the cost of weed control has been significantly reduced by promoting herbicide-resistant crops. However, with the large-scale promotion of herbicide-resistant crops and the long-term use of single herbicides, environmental safety problems such as weed resistance and escape of resistant genes have gradually been discovered. Currently, the development of functional genomics, bioinformatics and genetic engineering technology (especially the widespread application of gene editing technology in plants) has created conditions for the creation of herbicide-resistant crops and new efficient weed control systems. In this article, the main target genes of herbicides that inhibit amino acid biosynthesis, lipid metabolism, carotenoid, plastoquinone and tocopherol biosynthesis pathways and their action mechanisms are introduced at first. Secondly, two methods for mining new herbicide resistance genes and herbicide systems are introduced, including the directed mutation method of herbicide resistance genes within crops based on CRISPR/Cas system and the resistance gene guidance method based on the co-evolution theory of natural product and organisms in nature. Moreover, the research progress of three breeding methods for herbicide resistant crops was reviewed, including conventional breeding, transgenic breeding and CRISPR/Cas genome editing based breeding. Among them, the research progress of CIRSPR/Cas system, base editing technology, and prime editing system in cultivating herbicide resistant crops were highlighted. The main challenge faced by chemical control of weeds and herbicide resistant crops is resistant weeds and environmental safety issues, and gene escape, respectively. At present, the rapid development of genome editing technology provides new solutions and new opportunities for the development of herbicide resistant crops in the post genome era. Finally, the prospects for the future of herbicide-resistant crops were provided.
|
|
Table and Figures丨
Reference丨
Related Articles丨
Metrics
|
|
