WO2023093848A1 - 一种转基因抗虫耐除草剂玉米及其培育方法 - Google Patents

一种转基因抗虫耐除草剂玉米及其培育方法 Download PDF

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WO2023093848A1
WO2023093848A1 PCT/CN2022/134348 CN2022134348W WO2023093848A1 WO 2023093848 A1 WO2023093848 A1 WO 2023093848A1 CN 2022134348 W CN2022134348 W CN 2022134348W WO 2023093848 A1 WO2023093848 A1 WO 2023093848A1
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resistance
corn
resistant
insect
gene
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王磊
邹俊杰
罗彦忠
杨德志
张兰
刘源
郑红艳
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中国农业科学院生物技术研究所
北京博艾远上生物科技有限公司
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Definitions

  • the invention relates to the field of plant biotechnology, in particular to a transgenic insect-resistant and herbicide-resistant corn and a breeding method thereof.
  • Corn is the largest food crop in my country, and it is also an important feed and industrial raw material. It plays a very important role in national food security and national economic development.
  • corn borer the corn borer, whose adult insect belongs to Lepidoptera Pyralididae, is a world-wide moth-eating pest and an important factor affecting the yield and quality of corn.
  • the annual loss caused by insect pests in the world accounts for about 30% of the crop output. More than 15%.
  • the corn borer can damage all parts of the corn plant above the ground, making the damaged part lose its function and reducing the grain yield.
  • the current control measures are mainly to use a large amount of chemical pesticides, which not only seriously affects the ecological environment and biodiversity, increases production costs and labor intensity, but also increases the chance of human poisoning.
  • Spodoptera frugiperda is an important agricultural pest for global warning by the Food and Agriculture Organization of the United Nations (FAO). It harms more than 80 species of plants such as corn, rice, and tomato.
  • the corn armyworm eats corn leaves with its larvae, and when it occurs seriously, it will eat up the leaves in a short period of time, resulting in reduced production or even no harvest. Symptoms of damage are mainly larvae biting leaves.
  • the harm of cotton bollworm on corn is increasing year by year, and it has become an important pest affecting the yield and quality of corn.
  • Cotton bollworm larvae mainly feed on corn kernels. The newly hatched larvae concentrate on the top of the ear of corn and bite the filaments.
  • the current control measures are mainly the extensive use of chemical pesticides.
  • Bt insect resistance genes from Bacillus thuringiensis, which can be divided into different types according to different classification basis.
  • Hofte and Whiteley named the gene encoding the insecticidal crystal protein as the Cry gene, which was represented by a combination of Roman numerals I, II, III, IV, V and English letters.
  • Cry I has the best anti-insect effect on Lepidoptera insects
  • Cry II mainly resists Lepidoptera and Diptera insects
  • Cry III has better effect on Coleoptera insects
  • CryIV insect resistance is mainly against Diptera insects
  • Cry V is toxic to both Lepidoptera and Coleoptera insects.
  • Cry1Ab and Cry1Ac genes have specific toxicity to corn borer and other Lepidoptera pests.
  • transgenic corn varieties are monogenic insect-resistant corn, among which insect-resistant genes Cry1Ab/Ac, Cry1F, and Cry2Ab are widely used.
  • Field and laboratory experiments in China have shown that the Cry1Ab gene-transferred corn can control the whole growth period of the Asian corn borer, and has an impact on the feeding of the Asian corn borer; bug effect.
  • insects exposed to high selective pressure for a long time will develop resistance to Bt.
  • Cry1Ab has a better insecticidal effect on cotton bollworm
  • Cry1F has a better insecticidal effect on cutworms, etc., but the insecticidal spectrum of the two is not completely the same.
  • insect-resistant genes used in my country's transgenic insect-resistant corn include Cry1Ab, Cry1Ah, Cry1Ie, etc., but most of them use single-gene insect-resistant methods.
  • Glyphosate is a systemic, broad-spectrum, organophosphine herbicide applied to foliage. It has the characteristics of high efficiency, low toxicity, and easy degradation. It can competitively inhibit the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway of plants and bacteria.
  • EPSPS is an enzyme in the pathway of aromatic amino acid synthesis, which reversibly catalyzes the condensation of S3P (Shikimate-3-phosphate) and PEP (Phosphoenopyrate) into EPSP and inorganic phosphine. Transferring the CP4EPSPS gene capable of resisting glyphosate herbicides into crops can make the transgenic crops resistant to glyphosate.
  • the present invention provides a method for cultivating transgenic corn resistant to insects and herbicides and the compound resistance to insect-resistant and herbicides obtained thereby GMO corn.
  • the present invention provides a synthetic gene tandem expression cassette, which comprises mCry1Ab, mCry1F and mCP4EPSPS genes.
  • the mCry1Ab gene sequence is shown in sequence 1
  • the mCry1F gene sequence is shown in sequence 2
  • the mCP4EPSPS gene sequence is shown in sequence 3.
  • Another aspect of the present invention provides a plant expression vector comprising the tandem expression cassettes of the above-mentioned genes.
  • the plant expression vector is pL5.
  • Another aspect of the present invention provides a host cell comprising the above-mentioned gene tandem expression cassette, or comprising the above-mentioned plant expression vector.
  • the host cell is Agrobacterium, more preferably Agrobacterium EHA105.
  • Another aspect of the present invention provides the application of the gene tandem expression cassette, the plant expression vector, or the host cell in improving insect resistance and herbicide tolerance of corn.
  • the insect resistance is resistance to corn borer, fall armyworm, armyworm, cotton bollworm and other Lepidoptera insects, and the herbicide resistance is glyphosate resistance.
  • Another aspect of the present invention provides a method for cultivating insect-resistant and herbicide-tolerant transgenic corn, comprising integrating the gene tandem expression cassette, the plant expression vector, or the host cell into a recipient corn material.
  • the insect resistance is resistance to corn borer, fall armyworm, armyworm, cotton bollworm and other Lepidoptera insects, and the herbicide resistance is glyphosate resistance.
  • Another aspect of the present invention provides a combination of primers for detecting insect-resistant and herbicide-tolerant transgenic corn, the nucleotide sequences of which are shown in sequences 11 and 12.
  • the transgenic corn comprises the gene tandem expression cassette described in the present invention, and the insect resistance is preferably resistance to Lepidoptera insects such as corn borer, fall armyworm, armyworm, cotton bollworm, etc., and is resistant to weeding.
  • the agent is preferably resistant to glyphosate.
  • Another aspect of the present invention provides a method for detecting insect-resistant and herbicide-tolerant transgenic corn, which uses the primer combination described in the present invention to detect the transgenic corn.
  • the transgenic corn comprises the gene tandem expression cassette described in the present invention, and the insect resistance is preferably resistance to Lepidoptera insects such as corn borer, fall armyworm, armyworm, cotton bollworm, etc., and is resistant to weeding.
  • the agent is preferably resistant to glyphosate.
  • the last aspect of the present invention provides a sequence for identifying an exogenous insert in insect-resistant and herbicide-tolerant transgenic maize, which includes the exogenous insert and its flanking sequences.
  • the sequence is shown in SEQ ID NO: 10.
  • the exogenous insertion fragment comprises the gene tandem expression cassette described in the present invention
  • the insect resistance is preferably resistance to Lepidoptera insects such as corn borer, fall armyworm, armyworm, cotton bollworm, etc.
  • the herbicide resistance is preferably glyphosate resistance.
  • the present invention significantly improves the resistance of transgenic corn to Lepidoptera insects such as corn borer, fall armyworm, armyworm, and cotton bollworm.
  • the resistance to glyphosate has also reached a high level of resistance.
  • Figure 1 Plasmid map of pL5.
  • RB right border sequence
  • pVS1-REF Agrobacterium replicon
  • ColE1 Escherichia coli replicon
  • Kan kanamycin resistance gene
  • T35s CaMV35S terminator
  • mCP4EPSPS glyphosate resistance gene
  • Ubi maize ubiquitin promoter
  • Thsp17 heat shock protein terminator
  • mCry1F insect resistance gene
  • CTP signal peptide
  • OsAct2 rice promoter
  • E35S Enhanced CaMV35S promoter
  • mCry1Ab insect resistance gene
  • Tnos Nos terminator
  • Figure 2 PCR detection of mCry1Ab, mCry1F, mCP4EPSPS genes in BFL5 strain.
  • M 100bp molecular weight standard
  • 1 mCry1F (BFL5-1);
  • Figure 3 BFL5 strain-specific PCR detection.
  • M 100bp molecular weight standard; 1: BFL5-1;
  • Figure 4 Gene expression levels of mCry1Ab, mCry1F, and mCP4EPSPS genes in BFL5 materials.
  • A The test results at the 4-5 leaf stage of corn; B: The test results at the mature stage of corn.
  • Example 1 Obtaining of transgenic materials
  • the Cry1Ab, Cry1F and CP4EPSPS genes were comprehensively optimized to remove AT-rich sequences such as ATTTA and AATTAA that affect mRNA stability, and to increase the GC content of the gene coding region. Sequences to improve translation efficiency were added before the codons to enable efficient expression and translation in maize.
  • the Cry1Ab, Cry1F and CP4EPSPS gene sequences were named mCry1Ab, mCry1F and mCP4EPSPS respectively according to the comprehensive codon optimization of maize codons.
  • the nucleotide sequences are as follows: Sequences 1-3 are shown.
  • the basic vector is pCAMBIA1300, and its selection marker in bacteria is Kan gene, which encodes aminoglycoside phosphotransferase, and there is no report that its encoded product has toxic side effects on animals. Since it is located outside the T-DNA region, it cannot be transferred into plant cells.
  • ColE1 comes from Escherichia coli, and its main function is to help the replication of vectors in Escherichia coli. It is widely used in the construction of engineering vectors. It only exists as the origin of replication and does not encode proteins. Moreover, it is located outside the T-DNA region and cannot be transferred into plant cells.
  • pVS1-REP comes from Pseudomonas, and its main function is to help the plasmid replicate in Agrobacterium. It is widely used in the construction of engineering vectors. Because it is located outside the T-DNA region, it cannot be transferred to plant cells.
  • T-DNA is derived from Ti plasmid, but oncogenic genes and sequences irrelevant to T-DNA transfer have been removed, and only the right border sequence (RB) and left border sequence (LB) necessary for T-DNA transfer are retained.
  • RB and LB are exonuclease recognition sequences during T-DNA transfer and do not encode any gene products.
  • the target genes mCP4EPSPS, mCry1F, mCry1Ab and their supporting promoters and terminators were inserted between RB and LB, and a plant expression vector was constructed according to conventional molecular biology methods, named pL5 plasmid, size: 17.8kb.
  • the carrier map is shown in Figure 1.
  • the expression cassettes of the target genes mCP4EPSPS, mCry1F, and mCry1Ab are shown in Table 1-3.
  • CTP Chloroplast transfer signal peptide mCP4EPSPS Encodes CP4EPSPS protein, resistant to glyphosate. Thsp17 Hsp17 terminator, terminates transcription.
  • mCry1F Encodes Cry1F protein, anti-lepidoptera insects.
  • T35S CaMV35S terminator terminates transcription.
  • mCry1Ab Encodes Cry1Ab protein, anti-Lepidoptera insects. Tnos Nos terminator, terminates transcription.
  • the target gene and other gene elements are introduced into the recipient corn material.
  • the transformation process is as follows: take corn young ears 9-12 days after pollination, peel off the immature embryos, place them in a liquid medium for infection, and use them for Agrobacterium infection.
  • Resistant calli were transferred to regeneration medium to obtain mature embryoid bodies. Put the embryoid bodies on MS medium to form seedlings and take root to obtain regenerated corn seedlings. And through the method of backcross breeding, the transformation event was backcrossed with the corn inbred line Zheng 58, and the insect-resistant and herbicide-resistant corn BFL5 with the genetic background of Zheng 58 containing the exogenous gene was obtained.
  • Example 2 Detection of genetic stability of target gene in transgenic material
  • Each gene was stably inherited in each generation of the transformant, and its segregation ratio conformed to the Mendelian single-site inheritance rule, indicating that the three target genes were only integrated at one site.
  • the PCR band map of the BFL5 target gene is shown in Figure 2.
  • the primers for the PCR of the target gene are shown in Table 4 to Table 6, and the PCR composition and reaction conditions are shown in Table 7.
  • Primer I Hsp
  • CP4 5'-gtcatgggtctcgtctctga-3' (sequence 5)
  • Example 3 BFL5 flanking sequence analysis and specific PCR detection
  • the 25 transgenic corn events obtained were first identified for glyphosate resistance, and 10 events had a resistance of more than 4 times.
  • the insect resistance of the 10 events was identified respectively, and 4 events were screened out to be effective against pests such as corn borer. It showed high resistance, and further used the whole genome resequencing method to preliminarily determine the integration of the target gene in the maize genome.
  • 1 event expression box had a sequence loss
  • 2 were inserted into the maize gene
  • the intergenic region of BFL5 (named BFL5), further research on BFL5 found that it has no effect on the growth and development of maize, and the expression frame is complete, the gene expression level is high, and the inheritance is stable.
  • the purpose was determined by BLAST analysis in the maize genome database
  • the integration position of the gene in the genome and the specific sequence on the Tnos side are shown in SEQ ID NO: 10.
  • the primer pair for Tnos specific PCR detection consists of the single-stranded DNA molecule shown in sequence 11 and the single-stranded DNA molecule shown in sequence 12, and the amplified sequence is shown in sequence 10.
  • Tnos specific amplification primers The sequence of Tnos specific amplification primers is:
  • Primer 1 (BFL5n): 5'-gcatggtccaagtccacctt-3'; (SEQ ID NO: 11)
  • Theoretical amplification size is 300bp.
  • the PCR products were subjected to agarose gel electrophoresis.
  • Example 5 BFL5 target protein assay method and its expression level detection in different tissues
  • the expressions of the target gene proteins Cry1Ab, Cry1F and CP4EPSPS in the root, stem, leaf and grain tissues of maize transformant BFL5 were analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA).
  • ELISA double-antibody sandwich enzyme-linked immunosorbent assay
  • the results of ELISA showed that the target proteins Cry1Ab, Cry1F and CP4EPSPS could be detected in different organ samples of maize transformant BFL5, and the content of the target protein in different organs varied greatly, with higher content in leaves and lower content in grains.
  • the expression levels of target proteins in different tissues are shown in Table 10.
  • ⁇ means the standard deviation
  • NA means the value is lower than the effective detection range of the standard curve, not detected.
  • Embodiment 6 BFL5 insect resistance test
  • the tested insects Asian corn borer, Armyworm, Cotton bollworm and Spodoptera frugiperda, were continuously reared on artificial feed indoors for multiple generations. During the feeding process, no contact with any Bt preparations and Bt insecticidal proteins. They were reared under the conditions of temperature 27 ⁇ 1°C, relative humidity 70%-80%, and photoperiod L16:D8h.
  • the transgenic corn materials used in the experiment were: BFL5 (including fully optimized mCry1Ab, mCry1F and mCP4EPSPS expression cassettes), BFL4 (including partially optimized Cry1Ab, Cry1F and CP4EPSPS expression cassettes), Cry1Ab, Cry1F single-gene control corn, parental Compare Zheng 58.
  • BFL5 including fully optimized mCry1Ab, mCry1F and mCP4EPSPS expression cassettes
  • BFL4 including partially optimized Cry1Ab, Cry1F and CP4EPSPS expression cassettes
  • Cry1Ab Cry1F single-gene control corn
  • parental Compare Zheng 58 parental Compare Zheng 58.
  • the corn materials were all grown in pots in the greenhouse.
  • Bioassay of corn borer, cotton bollworm, and Spodoptera frugiperda Take 4-6 corn plants at the 4-6 leaf stage and bring them back to the room, select the young heart leaves that have just unfolded, rinse them with tap water, and rinse them once with distilled water , use filter paper to absorb water droplets on the surface and then dry it. Cut the corn leaves from which the main veins have been removed into leaves with a length and width of about 1 cm, and put them into 24-well cell culture plates, with 1-2 pieces per well. One newly hatched larva for testing was inserted into each well. Each plate is 1 replicate, repeated 4 times. Place them under the conditions of 27 ⁇ 1°C, relative humidity 70%-80%, and photoperiod L16:D8h.
  • transgenic corn BFL5 The results of indoor insect resistance of transgenic corn BFL5 to O. corn borer showed that the newly hatched larvae of O. corn Borer all died after feeding on the leaves of the transgenic material BFL5 for 4 days; 17.7% survived, while the survival rate of the non-transgenic control Zheng 58 was about 80%, and there was a significant difference. It shows that the transgenic material BFL5 is significantly better than the single-gene transgenic material, and the transgenic maize BFL4 only partially optimizes the codons of Cry1Ab, Cry1F and CP4EPSPS, and the resistance of BFL5 to O. corn borer is better than that of BFL4.
  • transgenic corn BFL5 to armyworm The results of indoor insect resistance of transgenic corn BFL5 to armyworm showed that the newly hatched armyworm larvae all died after feeding on the transgenic material BFL5 leaves for 6 days, while most of them survived after 6 days of transgenic Cry1F single gene, and most of them survived after 6 days of transgenic Cry1Ab single gene. Survival, while the survival rate on the non-transgenic control Zheng 58 was over 90%, and there was a significant difference. It shows that the resistance of BFL5 to armyworm is significantly better than that of transgenic materials, and is obviously better than that of BFL4.
  • Embodiment 7 BFL5 field anti-glyphosate test
  • Transgenic maize BFL5 (hereinafter referred to as BFL5), and the corresponding non-transgenic maize control Zheng 58.
  • the target herbicide is Roundup (41% glyphosate isopropylamine salt) produced by Monsanto Company.
  • Randomized block design with 3 repetitions. There is a 1.0m wide isolation zone between the residential areas, and the area of the residential area is 24m 2 .
  • transgenic corn was not sprayed with herbicide
  • transgenic corn was sprayed with target herbicide glyphosate
  • corresponding non-transgenic corn was not sprayed with herbicide
  • Level 3 Moderate phytotoxicity of corn, which can recover in the future without affecting the yield
  • Dose gradient of herbicide application the medium dose, 2 times and 4 times of the medium dose on the pesticide registration label are 900g a.i./ha, 1800g a.i./ha, 3600g a.i./ha, respectively.
  • Application method stem and leaf treatment, spray volume 450L/ha.
  • the seedling rate of BFL5 was 100% after 1, 2 and 4 weeks of treatment without spraying and spraying three concentrations of glyphosate, such as 900g a.i./ha, 1800g a.i./ha, 3600g a.i./ha.
  • the seedling rate of the non-transgenic corn control was 100% after 1, 2 and 4 weeks of treatment without spraying. After spraying 900, 1800 and 3600g

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Abstract

提供了一种转基因抗虫耐除草剂玉米及其培育方法。该通过将基因mCry1Ab、mCry1F和mCP4EPSPS组合为基因串联表达框的形式，整合到受体玉米材料中，从而与现有技术中采用的单基因抗虫或耐除草剂基因相比，提高了转基因玉米对鳞翅目昆虫的抗性，对草甘膦的耐性也达到了高抗的水平。

Description

一种转基因抗虫耐除草剂玉米及其培育方法 技术领域
本发明涉及植物生物技术领域，具体涉及一种转基因抗虫耐除草剂玉米及其培育方法。
背景技术
玉米是我国第一大粮食作物，也是重要的饲料及工业原料，在国家粮食安全和国民经济发展中占有非常重要的地位。
在玉米生长发育过程中，常遭受病害、虫害、草害的影响。世界范围内玉米害虫达350多种之多，其中以蛀茎性和食叶性的欧洲玉米螟和亚洲玉米螟最为严重。玉米螟俗名钻心虫，成虫属鳞翅目螟蛾科(Lepidoptera Pyralididae)，为世界性的蛀食性大害虫，是影响玉米产量和品质的重要因素，世界每年因虫害造成的损失约占农作物产量的15％以上。玉米螟可危害玉米植株地上的各个部位，使受害部分丧失功能，降低籽粒产量。目前的防治措施主要是大量使用化学杀虫剂，这不仅严重影响了生态环境和生物多样性，增加了生产成本和劳动强度，也加大了人体中毒几率。草地贪夜蛾是联合国粮农组织(FAO)全球预警的重要农业害虫，为害玉米、水稻、番茄等超过80种植物，具有危害大、繁殖能力强、迁飞速度快、食量大等特点。玉米粘虫以幼虫暴食玉米叶片，严重发生时，短期内吃光叶片，造成减产甚至绝收。为害症状主要以幼虫咬食叶片。棉铃虫在玉米上危害逐年加重，已成为影响玉米产量和品质的一种重要害虫。棉铃虫幼虫主要取食玉米籽粒。初孵幼虫集中在玉米果穗顶部咬食花丝。目前的防治措施主要是大量使用化学杀虫剂。
来自苏云金芽孢杆菌的Bt抗虫基因种类繁多，根据不同的分类依据可将其分为不同类型。在1989年，Hofte和Whiteley将编码杀虫晶体蛋白基因称为Cry基因，同时用罗马数字I、II、III、IV、V和英文字母结合起来表示。Bt蛋白基因有5大类、14个亚类。Cry I类对鳞翅目昆虫抗虫效果最佳，Cry II类主要抗鳞翅目和双翅目昆虫，Cry III类对鞘翅目昆虫效果较好，CryIV抗虫性主要针对双翅目昆虫，Cry V类对鳞翅目和鞘翅目昆虫均有毒性。Cry1Ab、Cry1Ac基因对玉米螟等鳞翅目害虫具有专一性毒性。
当前推广利用的转基因玉米品种大部分是单基因抗虫玉米，其中抗虫基因Cry1Ab/Ac，Cry1F，Cry2Ab等应用较多。国内的田间和室内实验表明：转Cry1Ab基因玉米可对亚洲玉米螟实现全生育期防治，并对亚洲玉米螟的取食有影响；转Cry1F基因玉米人工饲料对亚洲玉米螟幼虫有较高的杀虫效果。然而随着转基因玉米的连年大面积种植，昆虫长期暴露于高的选择压下，将会对Bt产生抗性。已有研究表明实验室内全生育期汰选27代的亚洲玉米螟种群对Cry1Ab的抗性提高了100多倍；全生育期汰选37代的欧洲玉米螟种群对Cry1F的相对抗性水平达到几百倍，但目前还未发现对Cry1Ab和Cry1F有交互抗性的玉米螟。此外，Cry1Ab对棉铃虫具有较好的杀虫作用，Cry1F对地老虎等具有较好的杀虫作用，二者杀虫谱不完全相同。目前我国转基因抗虫玉米采用的抗虫基因包括Cry1Ab、Cry1Ah、Cry1Ie等，但大多采用单基因抗虫方式。
草甘膦(Glyphosate)是一种内吸性、广谱性、施用于叶面的有机膦类除草剂，具有高效、低毒、易降解等特点。其可以竞争性抑制植物和细菌的莽草酸途径中5-烯醇丙酮莽草酸-3-磷酸合成酶(EPSPS)的活性。EPSPS是芳香族氨基酸合成途径中的一个酶，它可逆地催化S3P(Shikimate-3-phosphate)和PEP(Phosphoenopyrate)缩合成EPSP和无机膦。将具有抗草甘膦除草剂能力的CP4EPSPS基因转入农作物中，可使转基因作物具有抗草甘膦能力。
鉴于目前我国转基因抗虫玉米采用的多为单基因抗虫，且没有将Cry1Ab和Cry1F基因联合串联表达抗虫的研究，并同时与耐除草剂基因CP4EPSPS联用。因此，生产中亟需一种具有复合抗虫兼耐除草剂的转基因作物及其培育方法。
发明内容
本发明针对生产中亟需一种具有抗虫耐除草剂复合抗性的转基因玉米，提供了一种培育转基因抗虫耐除草剂玉米的方法以及由此获得的抗虫耐除草剂复合抗性的转基因玉米。
为此，本发明一方面提供了一种合成的基因串联表达框，其包含mCry1Ab、mCry1F和mCP4EPSPS基因。
在本发明优选的实施方案中，mCry1Ab基因序列如序列1所示，mCry1F基因序列如序列2所示，mCP4EPSPS基因序列如序列3所示。
本发明另一方面提供了包含上述基因串联表达框的植物表达载体。
在本发明优选的实施方案中，所述植物表达载体为pL5。
本发明另一方面提供了包含上述基因串联表达框、或包含上述植物表达载体的宿主细胞。
在本发明优选的实施方案中，所述宿主细胞为农杆菌，更加优选为农杆菌EHA105。
本发明另一方面提供了所述基因串联表达框、所述植物表达载体、或所述宿主细胞在提高玉米抗虫和耐除草剂特性上的应用。
在本发明优选的实施方案中，所述抗虫为抗玉米螟、草地贪夜蛾、黏虫、棉铃虫等鳞翅目昆虫，耐除草剂为耐草甘膦。
本发明另一方面提供了一种培育抗虫耐除草剂转基因玉米的方法，包括将所述基因串联表达框、所述植物表达载体、或所述宿主细胞整合到受体玉米材料中。
在本发明优选的实施方案中，所述抗虫为抗玉米螟、草地贪夜蛾、黏虫、棉铃虫等鳞翅目昆虫，耐除草剂为耐草甘膦。
本发明另一方面提供了一种检测抗虫耐除草剂转基因玉米的引物组合，其核苷酸序列如序列11和12所示。
在本发明优选的实施方案中，所述转基因玉米包含本发明所述的基因串联表达框，抗虫优选为抗玉米螟、草地贪夜蛾、黏虫、棉铃虫等鳞翅目昆虫，耐除草剂优选为耐草甘膦。
本发明另一方面提供了一种检测抗虫耐除草剂转基因玉米的方法，其采用本发明所述的引物组合对转基因玉米进行检测。
在本发明优选的实施方案中，所述转基因玉米包含本发明所述的基因串联表达框，抗虫优选为抗玉米螟、草地贪夜蛾、黏虫、棉铃虫等鳞翅目昆虫，耐除草剂优选为耐草甘膦。
本发明最后一方面提供了一种用于鉴定抗虫耐除草剂转基因玉米的外源插入片段的序列，其包含外源插入片段及其旁侧序列，所述序列如序列10所示。
在本发明优选的实施方案中，所述的外源插入片段包含本发明所述的基因串联表达框，抗虫优选为抗玉米螟、草地贪夜蛾、黏虫、棉铃虫等鳞翅目昆虫，耐除草剂优选为耐草甘膦。
由上述描述可知，与现有技术中采用的单基因抗虫或耐除草剂基因相比，本发明显著提高了转基因玉米对玉米螟、草地贪夜蛾、黏虫、棉铃虫等鳞翅目昆虫的抗性，对草甘膦的耐性也达到了高抗的水平。
附图说明
图1：pL5质粒图谱。
RB：右边界序列；pVS1-REF：农杆菌复制子；
ColE1：大肠杆菌复制子；Kan：卡那霉素抗性基因；
LB：左边界序列；T35s：CaMV35S终止子；
mCP4EPSPS：抗草甘膦基因；Ubi：玉米泛素启动子；
Thsp17：热激蛋白终止子；mCry1F：抗虫基因；
CTP：信号肽；OsAct2：水稻启动子；
E35S：增强型CaMV35S启动子；
mCry1Ab：抗虫基因；Tnos：Nos终止子。
图2：mCry1Ab、mCry1F、mCP4EPSPS基因在BFL5株系中PCR检测。
M：100bp分子量标准；1：mCry1F(BFL5-1)；
2：mCP4EPSPS(BFL5-1)；3：mCry1Ab(BFL5-1)；
4：mCry1F(BFL5-2)；5：mCP4EPSPS(BFL5-2)；
6：mCry1Ab(BFL5-2)；7：mCry1F(质粒，阳性对照)；
8：mCP4EPSPS(质粒，阳性对照)；9：mCry1Ab(质粒，阳性对照)；
10：mCry1Ab(郑58，阴性对照)；11：mCry1F(郑58，阴性对照)；
12：mCP4EPSPS(郑58，阴性对照)。
图3：BFL5株系特异性PCR检测。
M：100bp分子量标准；1：BFL5-1；
2：BFL5-2；3：郑58:(阴性对照)；
图4：mCry1Ab、mCry1F、mCP4EPSPS基因在BFL5材料中的基因表达量。
A：在玉米4-5叶期检测结果；B：在玉米成熟期检测结果。
具体实施方式
下面通过实施例对本发明作进一步的详细说明，旨在用于说明本发明而非限定本发明。应当指出，对于本领域技术人员而言，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也同样落入本发明的保护范围之内。
实施例1：转基因材料的获得
1、密码子优化
根据玉米的密码子使用频率对Cry1Ab、Cry1F和CP4EPSPS基因进行全面的密码子优化，去除影响mRNA稳定性的序列如ATTTA、AATTAA等富含AT序列，提高基因编码区GC含量，同时在起始密码子前添加提高翻译效率的序列等，使其在玉米中能够高效表达和翻译，依据玉米密码子全面优化后的Cry1Ab、Cry1F和CP4EPSPS基因序列分别命名为mCry1Ab、mCry1F和mCP4EPSPS，核苷酸序列如序列1-3所示。
2、表达载体构建
基础载体为pCAMBIA1300，其在细菌中的选择标记是Kan基因，其编码氨基糖苷磷酸转移酶，目前尚没有报道认为其编码产物对动物有毒副作用。由于其位于T-DNA区域外围,因此不能转移至植物细胞中。
ColE1来自于大肠杆菌，主要作用是帮助载体在大肠菌中的复制，被广泛应用于工程载体的构建，其只是作为复制起点存在，没有编码蛋白。而且其位于T-DNA区域外围,不能转移至植物细胞中。
pVS1-REP来自于假单胞杆菌，主要作用是帮助质粒在农杆菌中进行复制，被广泛应用于工程载体的构建，由于其位于T-DNA区域外围，因此不能转移至植物细胞中。
T-DNA来源于Ti质粒，但已去除了致瘤基因和与T-DNA转移无关的序列，只保留了T-DNA转移所必须的右边界序列(RB)和左边界序列(LB)。RB和LB为T-DNA转移时核酸外切酶识别序列，不编码任何基因产物。
目的基因mCP4EPSPS、mCry1F、mCry1Ab及其配套启动子和终止子插入到RB和LB之间，按照常规分子生物学方法构建了植物表达载体，命名为pL5质粒，大小：17.8kb。
载体图谱如附图1所示。目的基因mCP4EPSPS、mCry1F、mCry1Ab的表达框如表1-3所示。
表1 mCP4EPSPS表达框
名称 功能
OsAct2 水稻肌动蛋白基因启动子。
CTP 叶绿体转移信号肽
mCP4EPSPS 编码CP4EPSPS蛋白，抗草甘膦。
Thsp17 Hsp17终止子，终止转录。
表2 mCry1F表达框
名称 功能
Ubi 玉米泛素蛋白基因启动子。
mCry1F 编码Cry1F蛋白质，抗鳞翅目昆虫。
T35S CaMV35S终止子，终止转录。
表3 mCry1Ab表达框
名称 功能
E35S 增强型35S启动子。
mCry1Ab 编码Cry1Ab蛋白质，抗鳞翅目昆虫。
Tnos Nos终止子，终止转录。
3、转基因材料获得
采用农杆菌介导的方法，将目的基因及其它基因元件导入受体玉米材料中。
转化过程如下：取授粉9-12天的玉米幼穗，剥离幼胚，置于侵染用液体培养基，用于农杆菌侵染。将含有目的基因的质粒转化入农杆菌EHA105菌株，待OD 600＝0.3-0.4时，用侵染液重悬农杆菌，将玉米幼胚材料放入侵染液，进行农杆菌侵染。培养基中加入草甘膦作为选择压力，对被转化的材料进行筛选培养，每两周继代一次。经过2-3月的选择培养后，抗性愈伤组织在选择性培养基上生长迅速，颜色新鲜。将抗性愈伤组织转到再生培养基上，得到成熟的胚状体。将胚状体放到MS培养基上成苗和生根，得到再生玉米苗。并通过回交转育的方法，将转化事件与玉米自交系郑58进行回交，获得含有外源基因的郑58遗传背景的抗虫耐除草剂玉米BFL5。
实施例2：转基因材料中目的基因遗传稳定性检测
利用PCR技术分析目的基因mCry1Ab、mCry1F、mCP4EPSPS在玉米转化体BFL5中的整合情况及遗传稳定性。
结果表明，目的基因mCry1Ab、mCry1F、mCP4EPSPS在BC4F1、BC5F1、BC6F1代检测群体中均能检出，阳性特异片段大小分别与预期片段大小一致，且只有一条带。各基因在转化体各代间稳定遗传，其分离比符合孟德尔单位点遗传规律，说明三个目标基因中只整合在了一个位点。
BFL5目的基因PCR泳带图谱如附图2所示。
目的基因PCR的引物如表4至表6所示，PCR组成及反应条件如表7所示。
表4 mCP4EPSPS特异引物
Primer I(Hsp) 5'-tcgcacacacatcaaccaaa-3'(序列4)
Primer II(CP4) 5'-gtcatgggtctcgtctctga-3'(序列5)
表5 mCry1F特异引物
Primer I(C1F) 5'-tgagccagagcagcttcacg-3'(序列6)
Primer II(35S) 5'-catactaagggtttcttatatg-3'(序列7)
表6 mCry1Ab特异引物
Primer I(Nos) 5'-cccatctcataaataacgtc-3'(序列8)
Primer II(C1A) 5'-gccaagcacgagaccgtcaa-3'(序列9)
表7 PCR检测反应体系及反应条件
Figure PCTCN2022134348-appb-000001
实施例3：BFL5旁侧序列分析及特异性PCR检测
对获得的25个转基因玉米事件首先进行草甘膦抗性鉴定，抗性达到4倍以上的有10个事件，对10个事件分别进行抗虫性鉴定，筛选出4个事件对玉米螟等害虫表现出高抗，进一步利用全基因组重测序方法初步确定了目的基因在玉米基因组中的整合情况，其中1个事件表达框有序列丢失，2个插入在玉米基因内部，1个整合在玉米染色体上的基因间区(命名为BFL5)，对BFL5进一步的研究发现其对玉米的生长发育没有影响，且表达框完整，基因表达量高，遗传稳定。
根据获得的BFL5插入片段旁侧的玉米基因组序列，并进一步用PCR扩增测序的方法获得了BFL5事件的插入位点附近的玉米基因组特异性序列，通过在玉米基因组数据库中的BLAST分析确定了目的基因在基因组中的整合位置，其Tnos一侧的特异性序列如序列10所示。Tnos端特异性PCR检测引物对由序列11所示的单链DNA分子和序列12所示的单链DNA分子组成，扩增的序列如序列10所示。
提取BFL5基因组DNA进行特异性PCR扩增。Tnos端特异性扩增引物序列为：
Primer I(BFL5n):5’-gcatggtccaagtccacctt-3’；(序列11)
Primer II(Tnos):5’-agagtcccgcaattatacat-3’；(序列12)
理论扩增大小为300bp。
PCR反应体系及反应条件如表8所示。
表8 PCR检测反应体系及反应条件
Figure PCTCN2022134348-appb-000002
反应结束后，PCR产物进行琼脂糖凝胶电泳。
结果表明，BFL5的不同转化株都能检测到清晰的、与预期大小片段一致的单一目的条带，表明检测系统正常，无非特异性扩增，回交转育受体郑58和空白对照没有扩增条带，PCR电泳结果见附图3。
实施例4：BFL5目的基因qRT-PCR检测
提取玉米BFL5植株4-5片叶时期的幼苗的根、茎、叶，以及成熟植株的根、茎、叶以及种子中的RNA。RNA的提取参照试剂盒SV Total RNA Isolation System(Promega,USA)方法进行、反转录参照试剂盒A3500-Reverse Transcription System(Promega,USA)方法进行。目的基因qRT-PCR检测用引物见表9。
表9 BFL5转化材料中外源基因qRT-PCR引物信息
引物名称 序列
mCry1Ab fw 5'-cttacactcgctaccagctc-3'(序列13)
mCry1Ab rv 5'-cgttcaggtcggtgcagccc-3'(序列14)
mCry1F fw 5'-cgacaggttcgagttgatcc-3'(序列15)
mCry1F rv 5'-cagaactcgtcagagaggca-3'(序列16)
mCP4EPSPS fw 5'-acgggctcaagctcaacggt-3'(序列17)
mCP4EPSPS rv 5'-gagttcgatcttagctccaa-3'(序列18)
zmActin1 fw 5'-caccttctacaacgagctccg-3'(序列19)
zmActin1 rv 5'-taatcaagggcaacgtaggca-3'(序列20)
采用SYBR Premix ExTaq TM(Code DRR041A,TAKARA)荧光定量试剂盒，PCR仪为Applied Biosystems(http://www.AppliedBiosystems.com)Prism 7500 analyzer。BFL5植株mCry1Ab、mCry1F和mCP4EPSPS基因的表达量见附图4。
实施例5：BFL5目的蛋白测定方法及其在不同组织的表达量检测
采用双抗夹心酶联免疫法(ELISA)，分析玉米转化体BFL5根、茎、叶和籽粒组织中目的基因蛋白Cry1Ab、Cry1F和CP4EPSPS表达。ELISA实验方法和分析方法按照EnviroLogix公司试剂盒说明书进行。
首先称量0.1g各组织样品，经液氮研磨后，将样品加入到1mL样品提取缓冲液，同时用2mL样品提取缓冲液稀释标准品。使用EnviroLogix公司生产的CP4EPSPS、Cry1Ab/Ac、Cry1F的ELISA试剂盒检测目的蛋白的表达量。通过酶标仪分析不同样品的吸光值，根据标准蛋白的浓度及光密度值读数，拟合出标准曲线方程，最后利用Microsoft Excel计算各器官中表达的蛋白占样品组织重量来表示(μg/g)。
ELISA检测结果表明，目的蛋白Cry1Ab、Cry1F和CP4EPSPS在玉米转化体BFL5的不同器官样品中均可检出，不同器官中目的蛋白含量变化较大，叶片中含量较高，籽粒含量较低。目的蛋白在不同组织中的表达量见表10。
表10目的蛋白在玉米转化体BFL5不同组织中的表达量
Figure PCTCN2022134348-appb-000003
注：±表示标准差；NA表示数值低于标准曲线有效检测范围，未检出。
实施例6：BFL5抗虫性试验
1、供试昆虫
供试昆虫亚洲玉米螟、黏虫、棉铃虫及草地贪夜蛾均为在室内人工饲料上连续饲养多代。饲养过程中，不接触任何的Bt制剂和Bt杀虫蛋白。在温度27±1℃，相对湿度70％～80％，光周期L16：D8h的条件下饲养。
2、供试Bt玉米
试验用转基因玉米材料分别为：BFL5(包含全面优化后的mCry1Ab、mCry1F及mCP4EPSPS表达框)、BFL4(包含部分优化的Cry1Ab、Cry1F及CP4EPSPS表达框)、分别转Cry1Ab、Cry1F单基因对照玉米、亲本对照郑58。玉米材料均为在温室花盆种植。
3、抗虫性试验
玉米螟、棉铃虫、草地贪夜蛾生测：取4-6叶期的玉米植株4-6株带回室内，选取刚展开的幼嫩心叶，用自来水冲洗干净，再用蒸馏水冲洗1遍，用滤纸吸去表面水珠后晾干。将剔除主叶脉的玉米叶片，裁剪为长和宽均为1cm左右大小的叶片，放入24孔细胞培养板，每孔1-2片。每孔接入供试初孵幼虫1头。每板为1个重复，重复4次。置于27±1℃，相对湿度70％～80％，光周期L16：D8h的条件下饲养。
黏虫生测：取4-6叶期的玉米植株4-6株带回室内，选取幼嫩心叶清洗晾干后放入小养虫盒内，每盒接入20头初孵幼虫。每盒为1个重复，重复4次。置于27±1℃，相对湿度70％-80％，光周期L16：D8h的条件下饲养。
每2天调查1次，根据叶片组织被取食消耗情况随时添加或更换相同来源的新鲜叶片，并记录存活幼虫数，试验观察6天。
采用统计分析软件对不同害虫取食不同玉米材料后的存活率进行方差分析。
4、数据统计与分析
(1)对亚洲玉米螟的室内抗虫性测定
转基因玉米BFL5对亚洲玉米螟的室内抗虫性结果表明，亚洲玉米螟初孵幼虫取食转基因材料BFL5叶片4d后全部死亡，转Cry1F单基因4天时，大部分死亡，转Cry1Ab单基因4天仍有17.7％存活，而在非转基因对照郑58上存活率达80％左右，存在显著性差异。说明转基因材料BFL5显著优于转单基因的材料，同时转基因玉米BFL4仅对Cry1Ab、Cry1F及CP4EPSPS密码子进行了部分优化，BFL5对亚洲玉米螟的抗性优于BFL4。
表11转基因玉米BFL5对亚洲玉米螟的室内抗虫性
Figure PCTCN2022134348-appb-000004
注：表中数据为平均值±标准差，同列数据后不同小写字母表示差异显著(P<0.05)。
(2)对黏虫幼虫的室内抗虫性测定
转基因玉米BFL5对黏虫的室内抗虫性结果表明，黏虫初孵幼虫取食转基因材料BFL5叶片6d后全部死亡，而转Cry1F单基因6天时，大部分存活，转Cry1Ab单基因6天大部分存活，而在非转基因对照郑58上存活率达90％以上，存在显著性差异。说明BFL5对黏虫的抗性显著优于转单基因的材料，并明显优于BFL4。
表12转基因玉米BFL5对黏虫的室内抗虫性
Figure PCTCN2022134348-appb-000005
注：表中数据为平均值±标准差，同列数据后不同小写字母表示差异显著(P<0.05)。
(3)对棉铃虫的室内抗虫性测定
转基因玉米BFL5对棉铃虫的室内抗虫性结果表明，棉铃虫初孵幼虫取食转基因材料BFL5叶片4天后全部死亡，而转Cry1F基因4天时，仍有37.5％存活，转Cry1Ab单基因4天仍有12.5％存活，非转基因对照郑58存活率为70.8％，存在显著性差异。说明BFL5对棉铃虫的抗性显著优于转单基因材料以及BFL4。
表13转基因玉米BFL5对棉铃虫的室内抗虫性
Figure PCTCN2022134348-appb-000006
Figure PCTCN2022134348-appb-000007
注：表中数据为平均值±标准差，同列数据后不同小写字母表示差异显著(P<0.05)。
(4)对草地贪夜蛾的室内抗虫性测定
转基因玉米BFL5对草地贪夜蛾的室内抗虫性结果表明，草地贪夜蛾初孵幼虫取食转基因材料BFL5叶片4天后已全部死亡，表现为高抗，而转Cry1F单基因6天时仍有75％存活，转Cry1Ab单基因6天，仍有59.4％存活，非转基因对照郑58在6天后存活率仍达90％以上，存在显著性差异。说明BFL5对草地贪夜蛾的抗性显著优于转单基因材料，明显好于BFL4。
表14转基因玉米BFL5对草地贪夜蛾的室内抗虫性
Figure PCTCN2022134348-appb-000008
注：表中数据为平均值±标准差，同列数据后不同小写字母表示差异显著(P<0.05)。
实施例7：BFL5田间抗草甘膦试验
1、试验材料
转基因玉米BFL5(以下称BFL5)、对应的非转基因玉米对照郑58。目标除草剂采用孟山都公司生产的农达(41％草甘膦异丙胺盐)。
2、试验设计
随机区组设计，3次重复。小区间设有1.0m宽隔离带，小区面积24m 2。
设以下处理：(1)转基因玉米不喷施除草剂；(2)转基因玉米喷施目标除草剂草甘膦；(3)对应的非转基因玉米不喷施除草剂；(4)对应的非转基因玉米喷施目标除草剂草甘膦。
除草剂的施用剂量设3个梯度：(1)农药登记标签的中剂量；(2)中剂量的2倍量；(3)中剂量的4倍量。
3、调查方法
分别在用药后1周、2周和4周调查和记录成苗率、植株高度(选取最高的5株)、药害症状(选取药害症状最轻的5株)，包括：生长抑制、褪绿、枯斑、畸形等。
药害症状分级按GB/T 19780.42执行，具体如下：
1级：玉米生长正常，无任何受害症状；
2级：玉米轻微药害，药害少于10％；
3级：玉米中等药害，以后能恢复，不影响产量；
4级：玉米药害较重，难以恢复，造成减产；
5级：玉米药害严重，不能恢复，造成明显减产或绝产。
除草剂受害率按下列公式计算：X＝[∑(N×S)/(T×M)]×100
其中：X为受害率，单位为百分数(％)；N为同级受害株数；S为级别数；T为总株数；M为最高级别。
4、除草剂施用剂量及方法
除草剂的施用剂量梯度：农药登记标签的中剂量、中剂量的2倍量和4倍量分别为900g a.i./ha、1800g a.i./ha、3600g a.i./ha。
施药方法：茎叶处理，喷液量450L/ha。
5、结果分析
BFL5不喷药及喷施900g a.i./ha、1800g a.i./ha、3600g a.i./ha等3个浓度草甘膦处理1、2和4周后成苗率均为100％。非转基因玉米对照不喷药处理1、2和4周后成苗率均为100％，在喷施900、1800和3600g
a.i./ha草甘膦1周后植株均不能成苗，成苗率为0。
表15供试玉米材料喷施草甘膦后成苗率(％)
Figure PCTCN2022134348-appb-000009
注：表中数值为3个小区的平均值，成苗率均为100％或0％，因此未进行显著性分析。
BFL5不喷药及喷施900g a.i./ha、1800g a.i./ha、3600g a.i./ha等3个浓度草甘膦处理1、2和4周后没有药害。非转基因玉米对照在喷施900g a.i./ha草甘膦1周后，受害率为98.67％，有个别植株仍然存活，喷药2周后植株全部死亡。非转基因玉米对照在喷施1800、3600g a.i./ha草甘膦1周后植株全部死亡，受害率均为100％。
表16供试玉米材料喷施草甘膦后受害率(％)
Figure PCTCN2022134348-appb-000010
注：表中数值为3个小区的平均值。受害率均分别为0％或100％，因此未进行显著性分析。

Claims (10)

  1. 一种合成的基因串联表达框，其包含mCry1Ab、mCry1F和mCP4EPSPS基因，其中mCry1Ab基因序列优选如序列1所示，mCry1F基因序列优选如序列2所示，mCP4EPSPS基因序列优选如序列3所示。
  2. 包含权利要求1所述的基因串联表达框的植物表达载体，其优选为表达载体pL5。
  3. 包含权利要求1所述的基因串联表达框、或包含权利要求2所述的植物表达载体的宿主细胞，其优选为农杆菌，更加优选为农杆菌EHA105。
  4. 权利要求1所述的基因串联表达框、权利要求2所述的植物表达载体、或权利要求3所述的宿主细胞在提高玉米抗虫和耐除草剂特性上的应用，其中抗虫优选为抗鳞翅目昆虫，更加优选为抗玉米螟、草地贪夜蛾、黏虫和棉铃虫，耐除草剂优选为耐草甘膦。
  5. 一种培育抗虫耐除草剂转基因玉米的方法，包括将权利要求1所述的基因串联表达框、权利要求2所述的植物表达载体、或权利要求3所述的宿主细胞整合到受体玉米材料中，其中抗虫优选为抗鳞翅目昆虫，更加优选为抗玉米螟、草地贪夜蛾、黏虫和棉铃虫，耐除草剂优选为耐草甘膦。
  6. 一种检测抗虫耐除草剂转基因玉米的引物组合，其核苷酸序列如序列11和12所示。
  7. 一种检测抗虫耐除草剂转基因玉米的方法，其采用权利要求6所述的引物组合对转基因玉米进行检测。
  8. 根据权利要求6所述的引物组合或权利要求7所述的方法，其中转基因玉米包含权利要求1所述的基因串联表达框，抗虫优选为抗鳞翅目昆虫， 更加优选为抗玉米螟、草地贪夜蛾、黏虫和棉铃虫，耐除草剂优选为耐草甘膦。
  9. 一种用于鉴定抗虫耐除草剂转基因玉米的外源插入片段的序列，其包含外源插入片段及其旁侧序列，所述序列如序列10所示。
  10. 根据权利要求9所述的序列，其中外源插入片段包含权利要求1所述的基因串联表达框，抗虫优选为抗鳞翅目昆虫，更加优选为抗玉米螟、草地贪夜蛾、黏虫和棉铃虫，耐除草剂优选为耐草甘膦。
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ZOU JUNJIE;XU MIAOYUN;ZHANG LAN;ZHENG HONGYAN;WANG LEI: "Molecular Characteristics and Functional Evaluation of Transgenic Maize BFL4-1 With Stacked Insect and Herbicide Resistance Traits", JOURNAL OF AGRICULTURAL SCIENCE AND TECHNOLOGY, 10 February 2021 (2021-02-10), pages 31 - 37, XP093068472, ISSN: 1008-0864, DOI: 10.13304/j.nykjdb.2020.0111 *

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* Cited by examiner, † Cited by third party
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CN116514936A (zh) * 2023-06-29 2023-08-01 莱肯生物科技(海南)有限公司 一种抗虫蛋白及其制备方法和应用
CN116514936B (zh) * 2023-06-29 2023-09-29 莱肯生物科技(海南)有限公司 一种抗虫蛋白及其制备方法和应用
CN117004626A (zh) * 2023-10-07 2023-11-07 莱肯生物科技(海南)有限公司 抗虫表达盒及其应用

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