WO2014187311A1 - 一种使植物花粉特异性失活的载体及其用途 - Google Patents

一种使植物花粉特异性失活的载体及其用途 Download PDF

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WO2014187311A1
WO2014187311A1 PCT/CN2014/077966 CN2014077966W WO2014187311A1 WO 2014187311 A1 WO2014187311 A1 WO 2014187311A1 CN 2014077966 W CN2014077966 W CN 2014077966W WO 2014187311 A1 WO2014187311 A1 WO 2014187311A1
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pollen
gene
barnase
transgenic
plants
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PCT/CN2014/077966
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English (en)
French (fr)
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刘东风
常振仪
卢启清
王承旭
周君莉
唐晓艳
邓兴旺
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深圳市作物分子设计育种研究院
深圳兴旺生物种业有限公司
未名兴旺系统作物设计前沿实验室（北京）有限公司
兴旺投资有限公司
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Priority to CN201480003076.8A priority Critical patent/CN104838002A/zh
Publication of WO2014187311A1 publication Critical patent/WO2014187311A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

Definitions

  • the invention belongs to the technical field of plant genetic engineering and plant breeding, and specifically relates to the expression of a barnase gene and a functional inhibitory gene barstar simultaneously into a plant cell, respectively, using a pollen-specific expression promoter and a constitutive promoter, respectively, and finally reaching the plant
  • the purpose of pollen cell inactivation can completely overcome the problem of toxic leakage when using Barnase gene to regulate cell activity.
  • Barnase is an extracellular RNase produced by Bacillus amyloliquefaciens. Its product is expressed in the form of a precursor. During processing and transportation, the N-terminus is excised by about 20 amino acids to become a mature enzyme with a total of 110 amino acids. barnase is highly toxic and its micro-expression in specific cells can cause cell death. In 1990, Mariani et al. combined the tobacco anther tapetum-specific promoter TA29, which has specific temporal and spatial expression characteristics, with the barnase gene of B. amyloliquefaciens to form a chimeric gene, which was mediated by Agrobacterium tumefaciens-mediated method.
  • the present invention has made various attempts to finally select a technique capable of inhibiting the activity of barnase protein and barnase simultaneously expressed in plant cells, thereby inhibiting barnase activity and preventing its toxicity leakage, and surprisingly It was found that barstar can be used as a barnase inhibitory gene, and when both are transformed into plants for expression, and driven by constitutive promoters and pollen-specific promoters, respectively, it can cause pollen-specific inactivation of transgenic plants without It can cause phenotypic changes to other tissues and organs of plants.
  • the present invention proposes a novel strategy for constructing a pollen-inactivated vector, on the one hand, the expression of the barnase gene is driven by a pollen-specific promoter, and on the other hand, the 35S-group-shaped promoter drives the barstar gene, thus, the barnase is pollen
  • the specific high expression can kill the male gametes and achieve the purpose of pollen abortion.
  • the leakage of barnase in other plant tissues other than pollen can be completely inhibited by barstar, so the other phenotypes of the plants are completely normal.
  • the invention better overcomes the problem of toxic leakage of the barnase gene, and more effectively realizes the application of the Barnase gene in the creation of a male sterile line of plants.
  • the object of the present invention is to provide a novel method for creating a male sterile breeding system. Since the promoter selected to drive barnase expression is a promoter specifically expressed in the late pollen, the transgenic line obtained by the method can be realized. 50% pollen inactivation ratio, further, using the constitutive promoter 35S to drive the barstar gene to be expressed simultaneously in plants, can effectively avoid the problem of barnase toxicity in non-target tissue expression leakage.
  • the barnase gene protein involved in the present invention is an extracellular RNase produced by Bacillus amyloliquefaciens, which has strong cytotoxicity, and its specific expression in cells causes cell death, and thus is Widely used in the field of the establishment of plant male sterile lines.
  • the present invention constructs a vector comprising two expression cassettes, wherein the pollen-specific initiation is selected in the first expression frame.
  • the driver which drives the barnase gene, is specifically expressed in pollen.
  • the vector also contains a second expression cassette, namely the barstar gene expression cassette driven by the 35S promoter.
  • the present invention is based on the high structure of barnase, the preference of B. amyloliquei codon, and the preference of dicot codons.
  • the nucleotide sequence of barnase was engineered.
  • the modified nucleotide sequence is shown in SEQ ID NO: 1 in the Sequence Listing, and the wild type sequence is shown in SEQ ID NO: 2.
  • the modified barnase sequences provided by the present invention have the advantage of being more efficiently expressed in plants, which are useful for regulating pollen fertility and establishing males in plants using their ribonuclease activity. Sexual sterility is very important.
  • the inventors have driven two nucleotide sequences encoding barnase and barstar, respectively, under the driving of a pollen-specific promoter and a 35S constitutive promoter, enabling simultaneous implementation of barnase and barstar in pollen.
  • the expression vector containing the above two expression cassettes is transformed into a plant, and the obtained transgenic plants can achieve 50% of pollen lethality or no activity, thereby achieving the purpose of regulating the pollen fertility of the plant.
  • a nucleotide fragment encoding barnase and barstar is obtained by a PCR method or a synthetic method, and the T vector is ligated and confirmed by sequencing;
  • a promoter and a constitutive promoter for pollen-specific expression were amplified by PCR, ligated to the T vector and confirmed by sequencing;
  • transforming plants specifically, preferably transforming Arabidopsis thaliana, rapeseed, rice, and the like;
  • the "promoter” of the present invention is a DNA sequence located in the upstream region of the 5' end of the structural gene, which activates the RNA polymerase to bind accurately to the template DNA and has the specificity of transcription initiation.
  • nucleotide sequence described in the present invention is an arrangement order of nucleotides in nucleic acids (DNA and RNA). In many cases, the nucleotide sequence determines the biological function of the nucleic acid, ie different sequences have different biological functions.
  • polyen-specific expression promoter means a promoter which can drive a target gene to be specifically expressed in a plant pollen grain but not expressed in other organs of the plant.
  • plant expression vector refers to any vector known in the art which is capable of constantly expressing a foreign gene in a plant cell, such as pCAMBIA1301, pBI121 and the like.
  • transformation refers to any plant transformation method known in the prior art capable of introducing a foreign gene into a plant cell or plant tissue, such as Agrobacterium-mediated method, etc.
  • the "transgenic plant” as used in the present invention refers to a plant individual integrated with a foreign gene obtained by a gene transfer technique.
  • a nucleotide sequence having a foreign gene is stably stabilized in the genome of a transformed plant or a transgenic plant, and the exogenous nucleotide sequence can be stably inherited to the next generation.
  • the present invention Compared with the prior art techniques for creating a male sterile line using barnase, the present invention has the advantage that only the cytotoxicity of the barnase is retained in the pollen, and the barnase is eliminated in the other tissue cells of the plant to eliminate the leakage activity of the barnase. Therefore, the problem of toxic leakage in the expression of barnase driven by the pollen-specific promoter is effectively avoided, and the problems of abnormal development of other tissues and organs, important agronomic traits are avoided, and the establishment of plant genetic engineering male sterile lines and heterosis Utilization is of great significance.
  • Figure 1 is a T-DNA region map of the expression vector pBnKP.
  • LB and RB are the left and right borders of T-DNA, respectively;
  • P1 is the pollen-specific promoter of the Arabidopsis genome
  • BrSZl is the synthetic barnase gene coding region
  • T1 is the terminator from the Arabidopsis Rbcs gene
  • P2 is the constitutive promoter 35S from the tobacco mosaic virus.
  • Promoter Pr2 is a synthetic barstar gene from bacteria
  • T2 is a 35S terminator
  • P3 is a seed-specific promoter from the Brassica genome
  • Pr3 is a RFP gene coding region from reef coral
  • T3 is a bacterial-derived Nos. Terminator.
  • Figure 2 is an observation of Alexander staining of pBnKP transgenic Arabidopsis pollen. On the left is the pollen of transgenic Arabidopsis, and on the right is the pollen of wild-type Arabidopsis.
  • Figure 3 shows the fluorescence separation of pBnKP transgenic Arabidopsis seeds.
  • Figure 4 is a T-DNA region map of the expression vector pBnOs. LB and RB are the left and right borders of T-DNA, respectively;
  • PG47 is a pollen-specific promoter of the maize genome
  • BrSZl is a synthetic barnase gene coding region
  • T1 is a terminator derived from the Arabidopsis Rbcs gene
  • P2 is a constitutive promoter 35S promoter derived from tobacco mosaic virus.
  • Pr2 is a synthetic barstar gene from bacteria
  • T2 is a 35S terminator
  • Ltp2 is a seed and callus-specific promoter from the barley genome
  • Pr3 is a RFP gene coding region from reef coral
  • T3 is a bacterial source. Nos terminator.
  • Figure 5 is an I 2 -KI staining observation of pBnOs transgenic rice pollen. The pollen on the left side is the pollen of wild type rice, and the right side B is the pollen of transgenic rice.
  • Figure 6 shows the fluorescence separation of pBnOs transgenic rice seeds.
  • Example 1 Construction of PBnKP vector for transformation of Arabidopsis thaliana and Brassica napus
  • the pBnKP vector shown in Figure 1 was constructed by assembling the various elements of the barnase and barstar expression cassettes.
  • the vector comprises three expression cassettes, wherein the first expression cassette is a barnase expression cassette consisting of a pollen-specific expression promoter P1 from the Arabidopsis genome, as shown in SEQ ID NO: 3 in the sequence listing, artificially synthesized
  • the barnase gene coding region BrSZl as shown in SEQ ID NO: 1 in the list, and the terminator T1 from the Arabidopsis Rbcs gene, as shown in SEQ ID NO: 4 in the Sequence Listing, constitutes the first expression cassette P1: : BrSZl::Tl ;
  • the second expression cassette is the barstar expression cassette, consisting of the constitutive promoter 35S promoter P2 from Tobacco mosaic virus, as shown in SEQ ID NO: 5 in the sequence listing, synthetically from The bacterial barstar gene Pr2, as shown in SEQ ID NO: 6 in the sequence
  • the plasmid PBnKP was transferred into Agrobacterium tumefaciens strain EHA105 by electroporation, and Arabidopsis thaliana was genetically transformed by Agrobacterium-mediated method of flowering, and 38 transgenic plants were obtained.
  • a specific transforming receptor material is Columbia type Arabidopsis thaliana.
  • Example 2 Analysis of the 38 single-copy transgenic Arabidopsis plants obtained in Example 2 revealed that there was no significant morphological difference between the transgenic plants and the wild type control plants, but the pollen fertility was significantly different. It indicated that the transgenic plants did not cause phenotypic variation of any other tissues except for the pollen phenotypic changes.
  • the method used was as follows: In the flowering stage of Arabidopsis thaliana, a single plant was randomly selected from the transgenic Arabidopsis plants and their wild-type control plants, each flower was taken from each plant, and one flower was taken from each flower, and placed on a glass slide. In the center, add 1 drop of 1% Alexander solution (95% ethanol 10 mL, 1% malachite green 5 mL, 5 g phenol, 1% acid fuchsin 5 mL, 1% orange red G 0.
  • Alexander solution 95% ethanol 10 mL, 1% malachite green 5 mL, 5 g phenol, 1% acid fuchsin 5 mL, 1% orange red G 0.
  • the pollen staining rate of transgenic Arabidopsis plants was analyzed. The results showed that the dark blue pollen of the control plants accounted for 98.6% ⁇ 100%.
  • normal fertile pollen (orange) and abortion The pollen (green) ratio is very close to 1:1, indicating that the transgenic plants of the constructed vector can produce equal amounts of pollen carrying the exogenous cytotoxic gene and pollen carrying the exogenous cytotoxic gene, ie the construct PBnKP makes the transgenic strain 50% of the pollen is inactivated.
  • the expected function due to the constitutive expression of barstar in transgenic plants, also inhibits the phenotype of other tissues and organs that may be caused by barnase leakage. That is, the intended goal of vector design was achieved in Arabidopsis.
  • the transgenic plants of the constructed pBnKP vector can produce equal amounts of pollen carrying the exogenous cytotoxic gene and pollen not carrying the exogenous cytotoxic gene, ie the construct pBnKP 50% of the transgenic pollen is inactivated, only non-transgenic pollen containing no cytotoxic gene is fertile, and pollination is completed.
  • the transgenic locus can only be inherited by female gametes and separated by 1:1, and finally all single copies In the seeds of the transgenic plants, the separation ratio of fluorescent and non-fluorescent seeds was 1:1. It is further demonstrated that the various elements of the vector provided by the present invention as a whole work well in Arabidopsis to achieve the desired pollen inactivation function and seed labeling function.
  • Example 5 Fluorescence analysis of seeds produced by crossing pollen from male transgenic Arabidopsis plants with male plants.
  • One of the transgenic Arabidopsis plants obtained from 38 single-copy transgenic pBnKP vectors was selected, and T1 seed was harvested.
  • the plasmid pBnKP was transferred into Agrobacterium tumefaciens strain EHA105 by electroporation, and the rapeseed was genetically transformed by Agrobacterium-mediated method of flowering, and 26 single-copy transgenic plants were obtained.
  • the specific transforming receptor material is Brassica napus L. No. 9 .
  • the method used was as follows: In the flowering stage of rapeseed, a single plant was randomly selected from the transgenic rapeseed plants and their wild-type control plants, each flower was taken from each plant, and one anther was taken from each flower, placed in the center of the slide, and dropped. A drop of 1% Alexander solution, release the pollen with tweezers and anatomic needle, cover the coverslip, observe under the microscope, count the number of pollen pollen and the total number of pollen, dye the orange-red for fertile pollen, green for defeat Breeding pollen.
  • the pollen staining rate of the transgenic rapeseed plants was analyzed. The results showed that the orange-red pollen of the control plants accounted for 98.6% ⁇ 100%.
  • normal fertile pollen oval-red
  • abortive pollen Green
  • the vector provided by the present invention as a whole can also achieve the expected pollen inactivation function in rapeseed, and does not cause phenotypic variation of other tissues and organs of the transgenic plants, and proves that the barnase gene acts as a cytotoxic gene in pollen.
  • the function due to the constitutive expression of barstar in transgenic plants, also inhibits the phenotype of other tissues and organs that may be caused by barnase leakage. That is, the expected goal of the carrier design is also achieved in the rapeseed.
  • the pBnOs vector shown in Figure 4 was constructed by assembling the various elements of the barnase and barstar expression cassettes.
  • the vector comprises three expression cassettes, wherein the first expression cassette is a barnase expression cassette consisting of a pollen-specific expression promoter PG47 from the maize genome, as shown in SEQ ID NO: 11 of the sequence listing, a synthetic barnase gene
  • the second expression cassette is the barstar expression cassette, consisting of the constitutive promoter 35S promoter P2 from tobacco mosaic virus, as shown in SEQ ID NO: 5 in the sequence listing, synthetic The barstar gene Pr2 from bacteria, as shown in SEQ ID NO: 6 in the Sequence Listing, and the 35S terminator T2, as shown in SEQ ID NO: 7 in the Sequence Listing, constitute the second expression cassette P2::Pr2::
  • the third expression cassette is an RFP expression cassette consisting of a seed from the barley genome and a callus-specific expression promoter Ltp2, as shown in SEQ ID NO: 12 of the sequence listing, from the artificially synthesized RFP gene coding region Pr3, such as SEQ ID NO in the sequence listing : 9 , and the bacterial-derived Nos terminator T3, as shown in SEQ ID NO: 10 in the Sequence Listing, constitutes the third expression cassette Ltp2::Pr3::T3.
  • the plant expression vector PBnOs was transferred into Agrobacterium tumefaciens AGL0 strain by heat shock method, and the rice embryogenic callus was infested with Agrobacterium. The cells were cultured for 2-3 days in dark, then subjected to two-step resistance screening, pre-differentiation, differentiation and rooting culture. In the same step, 42 strains of single-transformed PBnOs vector rice To plants were obtained.
  • Example 7 The analysis of 42 single-copy pBnOs vector rice materials obtained in Example 7 showed that there was no obvious morphological difference between the transgenic rice plants and the wild-type control rice plants, but the pollen fertility was significantly different. It indicated that the transgenic plants did not cause phenotypic variation of any other tissues except for the pollen fertility phenotype.
  • the method used is as follows: In the flowering stage of rice, a single plant is randomly selected from the transgenic rice plants and their wild-type control rice plants. Each plant takes a flower, and each flower takes an anther, placed in the center of the slide, and drops. Add a drop of 1% I2-IK solution, release the pollen with tweezers and anatomic needle, cover with a cover slip, observe under the microscope, count the number of pollen pollen and pollen, and dye the dark blue for fertile pollen. The pigmented aborted pollen (Fig. 5 shows the dyed fertile pollen grains and sterile pollen grains).
  • the pollen staining rate of transgenic rice plants was analyzed. The results showed that the dark blue pollen of the control plants accounted for 98.6% ⁇ 100%.
  • normal fertile pollen dark blue
  • abortive pollen Can not be colored
  • the ratio is very close to 1:1, indicating that the transgenic plants of the constructed vector can produce equal amounts of pollen carrying the exogenous cytotoxic gene and pollen carrying the exogenous cytotoxic gene, ie the construct pBnOs makes the transgenic line 50% of pollen is inactivated.
  • the vector provided by the present invention as a whole can achieve the expected pollen inactivation function in rice, and does not cause phenotypic variation of other tissues and organs of the transgenic plants, and proves that the barnase gene acts as a cytotoxic gene in pollen. Function, and due to the constitutive expression of barstar in transgenic plants, it also inhibits the phenotype of other tissues and organs that may be caused by barnase leakage. That is, the expected goal of vector design is achieved in rice.
  • the 42 single-copy transgenic rice plants transformed with pBnOs vector were harvested and the ratio of fluorescent to non-fluorescent seeds was determined.
  • the results showed that the seeds of these lines showed a 1:1 separation ratio (Fig. 6). ), that is, the seed carrying and not carrying the foreign gene is a 1:1 separation ratio, since the fluorescent gene Pr3 is linked to the cytotoxic gene BrSZl in the same T-DNA, indicating that the constructed transgenic plant of the pBnOs vector can Produce an equal amount of pollen carrying an exogenous cytotoxic gene and pollen that does not carry an exogenous cytotoxic gene, ie, the construct pBnOs inactivates 50% of the transgenic pollen, and only non-transgenic pollen without cytotoxic genes is fertile , and complete pollination, the transgenic site can only be inherited by female gametes and 1:1 separated, and finally the seed of all single-copy transgenic plants is solid, the ratio of fluorescence to non-fluorescent seed separation is 1:1. It is further demonstrated
  • Example 10 Fluorescence analysis of seeds produced by crossing pollen from male plants with transgenic rice plants: 1 strain of transgenic rice plants obtained from 42 single-copy to pBnOs vectors, after harvesting T1 seeds, fluorescent color selection , the partially sorted red fluorescent seeds are germinated, planted in stages, and in line with the rice sterile line. Longhua 916A is planted adjacent to each other. When the flowering period meets, the pollen of the plant with red fluorescent seeds is pollinated by the artificial powder to the rice sterile line Longhua 916A, and all the sterile lines Longhua 916A are further harvested. The sturdy seeds were subjected to fluorescence analysis. Among the more than 12,000 seeds harvested, no red fluorescent seeds were detected.

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Abstract

一种使植物花粉特异性失活的载体及其用途。通过将barnase基因与其功能抑制基因barstar同时转入植物细胞中表达，分别使用花粉特异性表达启动子和组成型启动子驱动，可以使植物花粉细胞失活，又可以抑制Barnase基因在其它组织器官中的毒性泄露。

Description

一种使植物花粉特异性失活的载体及其用途
技术领域
本发明属于植物基因工程和植物育种技术领域， 具体涉及通过将 barnase基因与其功能 抑制基因 barstar同时转入植物细胞中表达， 分别使用花粉特异性表达启动子和组成型启动 子驱动， 最终可以达到植物花粉细胞失活的目的， 同时又可以彻底克服利用 Barnase基因调 节细胞活性时的毒性泄露问题。
背景技术
Barnase是解淀粉芽孢杆菌 (Bacillus amyloliquefaciens)产生的一种胞外 RNA酶，其产物 以前体形式表达， 在加工、 运输过程中 N端被切除约 20个氨基酸， 成为成熟酶， 共有 110 个氨基酸。 Barnase 具有强烈的毒性，它在特定细胞中的微量表达就可以造成该细胞的死亡。 1990年， Mariani 等将具有特异时空表达特性的烟草花药绒毡层特异启动子 TA29， 与解淀 粉芽孢杆菌的 barnase基因结合， 构成嵌合基因， 用根癌农杆菌介导法， 将上述嵌合基因导 入: W草， 获得了转基因植物 (Mariani C等， Induction of male sterility in plants by a chimaeric ribonuclease gene, Nature, 1990, 347： 737-741 )。 结果， 有相当大一部分转基因植株花药 皱缩、 不散粉、 自交不能结实， 但雌蕊正常， 可由其它植株授粉结实； 组织切片未发现绒 毡层， 花粉囊变形， 无小孢子和花粉粒。 因此， 他们认为， TA29-barnaSe嵌合基因在植物细 胞中的特异表达， 选择性破坏了花的绒毡层， 从而阻止了花粉的形成， 导致雄性不育。
20年来， Barnase被导入油菜、 烟草、 棉花、 小麦、 玉米、 水稻等各种作物进行了雄性 不育诱导的研究。 在利用 barnase创建植物雄性不育系的过程中也发现了一系列的问题， 如 barnase蛋白的温度敏感性问题、 驱动其表达的花粉特异启动子的特异性不够或上游有 CaMV35S等强启动子而导致的毒性泄露问题等。 对于上游有 CaMV35S等强启动子而导致的 毒性泄露问题， 研究人员采取了在 barnase所在的表达框和上游强启动子所在的表达框之间 插入比较长的核酸序列的方法来减少上游强启动子对 barnase表达的时空特异性的影响， 从 而防止雄性不育植物其他组织器官中不良性状的产生 （Jagannath 等， The use of a Spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high-frequency generation of transgenic male sterile lines in Brassica juncea L. , Mol. Breeding, 2001 , 8: 11-23 )。
为了解决启动子特异性不够等原因导致的毒性泄露问题， 2002年， Burgess等将 Barnase 也分成了 N端（l-36aa)和 C端 （37-llOaa)两个片段， 分别由同一个启动子绒毡层特异启 动子 127a启动转化番茄。只含有 N端或 C端的转基因植物表现为雄性可育， 将两个转基因 植物杂交， 得到的含有 N端和 C端两个片段的转基因植物表现为雄性不育， 且其他表型完 全正常 (Burgess等， A novel, two-component system for cell lethality and its use engineering nuclear male- sterility in plants， Plant J， 2002， 31： 113-125)。
上述方法虽然一定程度或剂量上提高了 barnase基因表达的特异性、 减弱了 Barnase基 因的泄露对植物生长发育的不良影响，但并没有从根本上解决 barnase基因的毒性泄露问题。 针对这一问题， 本发明进行了多种尝试， 最终选择了采用能够抑制 barnase蛋白活性的基因 与 barnase同时在植物细胞中表达的技术思想， 来抑制 barnase活性， 防止其毒性泄露问题， 并惊奇地发现， barstar可以作为 barnase的抑制基因， 将二者同时转化进入植物中表达， 并分别用组成型启动子和花粉特异性启动子驱动时， 即可以导致含转基因的花粉特异失活， 同时又不会对植物其它组织器官造成表型改变的效果。 因此， 本发明提出了一种新的花粉 失活载体的构建策略，一方面 barnase基因的表达由花粉特异性启动子驱动，另一方面由 35S 组成型启动子驱动 barstar基因， 这样， barnase在花粉中特异性高表达， 可以致死雄配子， 从而达到花粉败育的目的， 同时， barnase在花粉以外的其它植物组织中的泄露， 就可以完 全被 barstar所彻底抑制， 因此植物的其他表型完全正常。本发明更好的克服了 Barnase基因 的毒性泄露问题， 更有效的实现了 Barnase基因在创制植物雄性不育系方面的应用。
发明内容
本发明的目的是提供一种新的创建雄性不育育种体系的方法， 由于所选驱动 barnase表 达的启动子为花粉后期特异表达启动子， 因此通过该方法所获得的转基因株系可以实现 50%的花粉失活比例， 进一步， 利用组成型启动子 35S驱动 barstar基因在植物中同时表达， 能够有效避免 barnase毒性在非靶标组织表达泄露的问题。
本发明所涉及的 barnase基因蛋白， 是解淀粉芽孢杆菌 (Bacillus amyloliquefaciens)产生 的一种胞外 RNA酶， 具有很强的细胞毒性， 它在细胞中的特异性表达会造成细胞的死亡， 因而被广泛应用于植物雄性不育系的创建等领域。
在本发明中， 为了解决 barnase基因毒性太大以及容易泄露， 易影响植物正常的生长发 育的问题， 本发明构建了包含两个表达框的载体， 其中第一表达框中选择了花粉特异性启 动子， 驱动 barnase基因在花粉中特异表达， 同时， 为了防止其在其它组织器官中泄露， 该 载体中还包含了第二表达框， 即由 35S启动子驱动的 barstar基因表达框。 该载体转入植物 细胞后， 在花粉中会同时表达两个蛋白， 由于 35S启动子在花粉细胞中活性较低， 而花粉 特异性启动子在花粉细胞中活性较高， 虽然 barstar对 barnase蛋白活性存在抑制作用， 但在 花粉中 barnase酶活剂量足可以导致所表达的花粉细胞的死亡。 而在花粉以外的组织器官， 存在少量的 barnase的表达造成毒性泄露，在这些组织器官中组成型高表达的 barstar则可以 完全抑制泄露的 barnase的蛋白活性， 保证在花粉以外的组织器官不造成非预期表型。 最终 克服 Barnase基因的毒性泄露问题， 更有效的实现了 Barnase基因在创制植物雄性不育系方 面的应用。
为了使得细菌来源的 barnase基因更好的在植物中， 尤其是在双子叶植物中表达， 本发 明根据 barnase的高级结构、 解淀粉芽孢杆菌密码子的偏好性、 双子叶植物密码子的偏好性 对 barnase的核苷酸序列进行改造。 改造后的核苷酸序列如序列表中 SEQ ID NO: 1所示， 野生型序列如 SEQ ID NO: 2所示。
与野生型的 barnase相比， 本发明所提供的改造后的 barnase序列具有在植物中能够更 有效的表达的优点， 这些优点对于利用其核糖核酸酶活性在植物中调控花粉育性及建立雄 性不育系是非常重要的。
在本发明的另一方面，发明人将分别编码 barnase和 barstar的两个核苷酸序列置于花粉 特异性启动子和 35S组成型启动子的驱动下，可以实现在花粉中 barnase和 barstar的同时空 的表达， 而在其它组织器官中则只有 barstar表达， 且 barnase只有少量泄露表达， 从而既达 到了本发明创制雄性不育系的目的， 又能克服 barnase基因即使在花粉特异启动子的驱动 下， 仍然存在其他组织器官中的微弱活性所导致的 barnase基因的毒性泄露问题。 将含有上 述两个表达框的表达载体转化植物， 获得的转基因植物中可以实现 50%的花粉致死或无活 力， 从而达到目标性调控植物花粉育性的目的。
本发明依次通过下列步骤实现：
( 1 ) 通过 PCR方法或人工合成方法得到编码 barnase和 barstar的核苷酸片段， 连接 T载 体并测序确认；
(2) 通过 PCR方法分别扩增得到花粉特异表达的启动子和组成型启动子， 连接 T载体并 测序确认；
( 3 ) 构建植物表达载体： 将编码 barnase和 barstar的核苷酸片段分别置于花粉特异表达的 启动子和组成型启动子的驱动下；
(4) 转化植物， 具体地， 优选转化拟南芥、 油菜和水稻等；
( 6)将获得的 TQ代转基因拟南芥、油菜和水稻的花粉进行活性染色分析。结果表明有 50% 花粉无活力， 从而达到目标性调控植物花粉育性的目的。
本发明所述的 "启动子"是一段位于结构基因 5'端上游区的 DNA序列， 能活化 RNA 聚合酶， 使之与模板 DNA准确地相结合并具有转录起始的特异性。
本发明所述的 "核苷酸序列"是核酸 (DNA和 RNA)中核苷酸的排列顺序。许多情况下， 核苷酸序列决定核酸的生物功能， 即不同序列有不同的生物功能。 本发明所述的 "花粉特异表达启动子"是指该启动子可以驱动目的基因在植物花粉粒 中特异表达而在植物的其它器官不表达的启动子。
本发明所述的 "植物表达载体"是指现有技术中已知的、 能够在植物细胞中恒定表达 外源基因的任何一种载体， 如 pCAMBIA1301， pBI121等。
本发明所述的 "转化"是指现有技术中已知的、 能够将外源基因导入植物细胞或植物 组织的任何一种植物转化方法， 如农杆菌介导法等
本发明所述的 "转基因植物"是指通过基因转移技术获得的整合有外源基因的植物个 体。 通常转化植物或转基因植物基因组中稳定带有外源基因的核苷酸序列， 可将该外源核 苷酸序列稳定地遗传给下一代。
本发明与现有的利用 barnase创建雄性不育系的技术相比， 其优势在于只有在花粉中保 留了 barnase的细胞毒性， 而在植物其它组织细胞中通过 barstar的抑制均消除了 barnase的 泄露活性， 从而有效避免了花粉特异启动子驱动 barnase表达时存在的毒性泄露问题， 同时 避免了其他组织器官发育异常、 重要农艺性状受到影响的问题， 对植物基因工程雄性不育 系的创建和杂种优势的利用都具有重要意义。 附图说明
图 1是表达载体 pBnKP的 T-DNA区图谱。 LB和 RB分别为 T-DNA的左边界和右边界；
P1为拟南芥基因组的花粉特异表达启动子， BrSZl为人工合成的 barnase基因编码区， T1 为来自于拟南芥 Rbcs基因的终止子， P2为来自于烟草花叶病毒的组成型启动子 35S启动子， Pr2为人工合成的来自于细菌的 barstar基因， T2为 35S终止子， P3为来自油菜基因组的 种子特异表达启动子， Pr3为来自礁珊瑚的 RFP基因编码区， T3为细菌来源的 Nos终止子。
图 2是 pBnKP转基因拟南芥花粉的 Alexander染色观察。 左侧为转基因拟南芥的花粉， 右侧为野生型拟南芥的花粉。 图 3 是 pBnKP转基因拟南芥种子的荧光分离情况。 图 4是表达载体 pBnOs的 T-DNA区图谱。 LB和 RB分别为 T-DNA的左边界和右边界；
PG47为玉米基因组的花粉特异表达启动子， BrSZl为人工合成的 barnase基因编码区， T1 为来自于拟南芥 Rbcs基因的终止子， P2为来自于烟草花叶病毒的组成型启动子 35S启动子， Pr2为人工合成的来自于细菌的 barstar基因， T2为 35S终止子， Ltp2为来自大麦基因组的 种子及愈伤特异表达启动子， Pr3为来自礁珊瑚的 RFP基因编码区， T3为细菌来源的 Nos 终止子。 图 5 是 pBnOs转基因水稻花粉的 I2-KI染色观察。左侧 Α为野生型水稻的花粉，右侧 B 为转基因水稻的花粉。 图 6是 pBnOs转基因水稻种子的荧光分离情况。
具体实 ϋ^Γ式
下述实施例中所用方法如无特别说明均为常规方法， 实验中所用的载体为
pCAMBIA1301。
实施例 1： 用于转化拟南芥和油菜的 PBnKP载体的构建
通过装配 barnase和 barstar表达框各个元件， 来构建图 1所示的 pBnKP载体。 该载 体含有 3个表达框， 其中， 第一表达框为 barnase的表达框， 组成为来自拟南芥基因组的 花粉特异表达启动子 Pl， 如序列表中 SEQ ID NO: 3所示， 人工合成的 barnase基因编码区 BrSZl , 如需列表中 SEQ ID NO: 1所示， 以及来自于拟南芥 Rbcs基因的终止子 Tl， 如序列 表中 SEQ ID NO: 4所示， 构成第一表达框 Pl:: BrSZl::Tl ; 第二表达框为 barstar表达框， 组成为来来自于烟草花叶病毒的组成型启动子 35S启动子 P2，如序列表中 SEQ ID NO: 5所 示， 人工合成的来自于细菌的 barstar基因 Pr2， 如序列表中 SEQ ID NO: 6所示， 以及 35S 终止子 T2， 如序列表中 SEQ ID NO: 7所示， 构成第二表达框 P2::Pr2::T2; 第三表达框为 RFP表达框， 组成为来自油菜基因组的种子特异表达启动子 P3， 如序列表中 SEQ ID NO: 8 所示， 来自人工合成的 RFP基因编码区 Pr3， 如序列表中 SEQ ID NO: 9所示， 以及细菌来 源的 Nos终止子 T3， 如序列表中 SEQ ID NO: 10所示， 构成第三表达框 P3::Pr3::T3。
将上述各表达框的元件测序验证后， 在 pCAMBIA1301载体中依次连入上述片段， 最 终得到植物表达载体 pBnKP, 如图 1所示。 实施例 2: 拟南 化
利用电激法将质粒 PBnKP转入农杆菌 EHA105菌株， 利用农杆菌介导的蘸花法对拟南芥 进行遗传转化， 得到转基因植株材料 38株。 具体的转化受体材料为哥伦比亚型拟南芥。
实施例 3: 转基因拟南芥植株的花粉育性检测
对实施例 2所得到的 38株单拷贝转基因拟南芥植株材料进行分析发现， 转基因植株和 野生型对照植株之间没有明显的形态差异， 但是花粉育性明显不同。 表明转基因植株除花 粉育性表型改变外， 没有造成任何其他组织器官的表型变异。
对 pBnKP构建体转化拟南芥得到的 38株单拷贝转基因材料， 进行花粉可染率检测， 同 时对野生型拟南芥进行花粉可染率检测 （图 2)。
采用的方法为： 在拟南芥开花期， 从转基因拟南芥植株及其野生型对照植株各随机抽 取单株， 各株取一朵花， 每朵花取 1个花药， 置于载玻片中央， 滴加一滴 1%的 Alexander 溶液 （95%乙醇 10mL， 1%孔雀石绿 5 mL， 5 g苯酚， 1%的酸性品红 5mL， 1%的橘红 G 0. 5 mL, 冰醋酸 2 mL, 丙三醇 25mL， 蒸熘水 50 mL)， 用镊子和解剖针释放花粉后， 盖上盖玻 片， 在显微镜下观察， 计数可染色花粉数和花粉总数， 染成橘红色的为可育花粉， 绿色的 为败育花粉 （图 2显示了染色后的可育花粉粒和不可育花粉粒）。
分析转基因拟南芥植株的花粉可染率， 结果显示对照植株的深蓝色花粉占 98. 6%〜 100%； 而多个随机抽取的转基因植株中， 正常可育花粉 （橘红色） 与败育花粉 （绿色） 比 例非常接近 1 : 1， 表明所构建的载体的转基因植株可以产生等量的携带外源细胞毒性基 因的花粉和不携带外源细胞毒性基因的花粉， 即构建体 PBnKP使转基因株系花粉的 50% 失活。 该结果表明本发明所提供的载体作为整体表达在拟南芥中可以达到预期的花粉失活 功能， 且没有造成转基因植株其他组织器官的表型变异， 证明 barnase基因作为细胞毒性 基因在花粉中发挥预期的功能， 而由于 barstar在转基因植株中的组成型表达， 同时抑制 了 barnase泄露造成可能的其他组织器官的表型。 即在拟南芥中达到了载体设计的预期目 标。
实施例 ： 转基因拟南芥植株的荧光种子与非荧光种子分离比分析
转化 pBnKP载体得到的 38株单拷贝转基因拟南芥植株收获种子后对其所结 T1代种子 进行荧光与非荧光种子的分离比例调查，结果表明这些株系的种子均显示 1： 1分离比（图 3)， 即携带和不携带外源基因的种子为 1 : 1分离比。 由于荧光基因 Pr3与细胞毒性基因 BrSZl 的表达框在同一个 T-DNA内连锁，表明所构建的 pBnKP载体的转基因植株可以产生等量的 携带外源细胞毒性基因的花粉和不携带外源细胞毒性基因的花粉， 即构建体 pBnKP使转 基因株系花粉的 50%失活， 只有不含细胞毒性基因的非转基因花粉可育， 并完成授粉， 转基 因位点只能够通过雌配子进行遗传并进行 1 : 1分离， 最终表现为所有单拷贝转基因植株所 结实的种子中， 荧光和非荧光种子分离比为 1 : 1。进一步证明本发明所提供的载体各元件作 为整体在拟南芥中工作良好， 达到预期的花粉失活功能和种子标记功能。
实施例 5：来自转基因拟南芥植株的花粉与雄性^ 植株杂交所产生的种子的荧光分析 从 38株单拷贝转 pBnKP载体得到的转基因拟南芥植株中任意选取 1株， 收获 T1代种 子后， 进行荧光色选， 将部分分选出来的红色荧光种子萌发后， 开花后授粉给拟南芥不育 系 Salk-152147， 进一步收获所有不育系 Salk_152147上所结实的种子， 进行荧光分析， 在 收获的 Salk-152147 5000多粒种子中， 没有检测到红色荧光种子， 证明 pBnKP载体的转基 因植株产生的可育花粉全部为不含有转基因的花粉， 进一步表明 pBnKP载体的转基因拟南 芥植株中所有含有细胞毒性基因 BrSZl的花粉， 全部为不育花粉， 实现了载体设计的花粉 失活功能。 实施例 6利用 pBnKP载体转化油菜实现调 基因油菜植株的花粉育性功能
利用电激法将质粒 pBnKP转入农杆菌 EHA105菌株， 利用农杆菌介导的蘸花法对油菜进 行遗传转化， 得到单拷贝转基因植株材料 26株。 具体的转化受体材料为甘蓝型油菜中双 9 号。
对所得到的 26株单拷贝转基因材料进行分析发现， 转基因植株和野生型对照植株之间 没有明显的形态差异， 但是花粉育性明显不同。 表明转基因植株除花粉育性表型改变外， 没有造成任何其他组织器官的表型变异。
对 pBnKP构建体转化油菜得到的 26株单拷贝转基因材料， 进行花粉可染率检测， 同时 对野生型油菜进行花粉可染率检测。
采用的方法为： 在油菜开花期， 从转基因油菜植株及其野生型对照植株各随机抽取单 株， 各株取一朵花， 每朵花取 1个花药， 置于载玻片中央， 滴加一滴 1%的 Alexander溶液， 用镊子和解剖针释放花粉后， 盖上盖玻片， 在显微镜下观察， 计数可染色花粉数和花粉总 数， 染成橘红色的为可育花粉， 绿色的为败育花粉。
分析转基因油菜植株的花粉可染率， 结果显示对照植株的橘红色花粉占 98. 6%〜100%； 而多个随机抽取的转基因植株中， 正常可育花粉 （橘红色） 与败育花粉 （绿色） 比例非常 接近 1 : 1， 表明所构建的载体的转基因植株可以产生等量的携带外源细胞毒性基因的花 粉和不携带外源细胞毒性基因的花粉， 即构建体 pBnKP使转基因株系花粉的 50%失活。 该 结果表明本发明所提供的载体作为整体表达在油菜中也可以达到预期的花粉失活功能， 且 没有造成转基因植株其他组织器官的表型变异， 证明 barnase基因作为细胞毒性基因在花 粉中发挥预期的功能， 而由于 barstar在转基因植株中的组成型表达， 同时抑制了 barnase 泄露造成可能的其他组织器官的表型。 即在油菜中也达到了载体设计的预期目标。
实施例 7 pBnOs载体构建及水稻转化：
通过装配 barnase和 barstar表达框各个元件， 来构建图 4所示的 pBnOs载体。 该载 体含有 3个表达框， 其中， 第一表达框为 barnase的表达框， 组成为来自玉米基因组的花 粉特异表达启动子 PG47, 如序列表中 SEQ ID NO: 11所示， 人工合成的 barnase基因编码区 BrSZl , 其核苷酸序列如列表中 SEQ ID NO: 1所示， 以及来自于拟南芥 Rbcs基因的终止子 T1，如序列表中 SEQ ID NO: 4所示，构成第一表达框 PG47:: BrSZl::Tl ;第二表达框为 barstar 表达框， 组成为来来自于烟草花叶病毒的组成型启动子 35S启动子 P2， 如序列表中 SEQ ID NO: 5所示， 人工合成的来自于细菌的 barstar基因 Pr2， 如序列表中 SEQ ID NO: 6所示， 以及 35S终止子 T2， 如序列表中 SEQ ID NO: 7所示， 构成第二表达框 P2::Pr2::T2; 第三表 达框为 RFP表达框， 组成为来自大麦基因组的种子及愈伤特异表达启动子 Ltp2， 如序列表 中 SEQ ID NO: 12所示， 来自人工合成的 RFP基因编码区 Pr3， 如序列表中 SEQ ID NO: 9 所示， 以及细菌来源的 Nos终止子 T3， 如序列表中 SEQ ID NO: 10所示， 构成第三表达框 Ltp2::Pr3::T3。
将上述各表达框的元件测序验证后， 在 pCAMBIA2300载体中依次连入上述片段， 最 终得到植物表达载体 pBnOs， 如图 4所示。
利用热激法将植物表达载体 PBnOs转入农杆菌 AGL0菌株， 用农杆菌侵染水稻胚性愈 伤， 暗中共培养 2-3天， 然后经过两步抗性筛选、 预分化、 分化和生根培养等步骤， 最终获 得单拷贝转 PBnOs载体水稻 To代植株 42株。
实施例 8转 pBnOs载体水稻植株的花粉育性检测
对实施例 7所得到的 42株单拷贝转 pBnOs载体水稻材料进行分析发现， 转基因水稻植 株和野生型对照水稻植株之间没有明显的形态差异， 但是花粉育性明显不同。 表明转基因 植株除花粉育性表型改变外， 没有造成任何其他组织器官的表型变异。
对 pBnOs构建体转化水稻得到的 42株单拷贝转基因材料， 进行花粉可染率检测， 同时 对野生型水稻植株进行花粉可染率检测 （图 5)。
采用的方法为： 在水稻开花期， 从转基因水稻植株及其野生型对照水稻植株各随机抽 取单株， 各株取一朵花， 每朵花取 1个花药， 置于载玻片中央， 滴加一滴 1%的 I2-IK溶液， 用镊子和解剖针释放花粉后， 盖上盖玻片， 在显微镜下观察， 计数可染色花粉数和花粉总 数， 染成深蓝色的为可育花粉， 不能着色的为败育花粉 （图 5显示了染色后的可育花粉粒 和不可育花粉粒）。
分析转基因水稻植株的花粉可染率， 结果显示对照植株的深蓝色花粉占 98. 6%〜100%； 而多个随机抽取的转基因植株中， 正常可育花粉 （深蓝色） 与败育花粉 （不能着色） 比例 非常接近 1 : 1， 表明所构建的载体的转基因植株可以产生等量的携带外源细胞毒性基因 的花粉和不携带外源细胞毒性基因的花粉，即构建体 pBnOs使转基因株系花粉的 50%失活。 该结果表明本发明所提供的载体作为整体表达在水稻中可以达到预期的花粉失活功能， 且 没有造成转基因植株其他组织器官的表型变异， 证明 barnase基因作为细胞毒性基因在花 粉中发挥预期的功能， 而由于 barstar在转基因植株中的组成型表达， 同时抑制了 barnase 泄露造成可能的其他组织器官的表型。 即在水稻中达到了载体设计的预期目标。
实施例 9转 pBnOs载体水稻植株的荧光种子与非荧光种子分离比分析
转化 pBnOs载体得到的 42株单拷贝转基因水稻植株收获种子后对其所结 T1代种子进 行荧光与非荧光种子的分离比例调查， 结果表明这些株系的种子均显示 1 : 1分离比（图 6)， 即携带和不携带外源基因的种子为 1 : 1分离比， 由于荧光基因 Pr3与细胞毒性基因 BrSZl 的表达框在同一个 T-DNA内连锁，表明所构建的 pBnOs载体的转基因植株可以产生等量的 携带外源细胞毒性基因的花粉和不携带外源细胞毒性基因的花粉， 即构建体 pBnOs使转 基因株系花粉的 50%失活， 只有不含细胞毒性基因的非转基因花粉可育， 并完成授粉， 转基 因位点只能够通过雌配子进行遗传并进行 1 : 1分离， 最终表现为所有单拷贝转基因植株所 结实的种子中， 荧光和非荧光种子分离比为 1 : 1。进一步证明本发明所提供的载体各元件作 为整体在水稻中工作良好， 达到预期的花粉失活功能和种子标记功能。
实施例 10： 来自转基因水稻植株的花粉与雄性 植株杂交所产生的种子的荧光分析 从 42株单拷贝转 pBnOs载体得到的转基因水稻植株中任意选取 1株， 收获 T1代种子 后， 进行荧光色选， 将部分分选出来的红色荧光种子萌发后， 分期播种， 并与水稻不育系 隆华 916A相邻种植， 待到二者花期相遇时， 通过人工赶粉， 将红色荧光种子发育的植株的 花粉， 授粉给水稻不育系隆华 916A， 进一步收获所有不育系隆华 916A上所结实的种子， 进 行荧光分析， 在收获的 12， 000多粒种子中， 没有检测到红色荧光种子， 证明 pBnOs载体的 转基因水稻植株产生的花粉全部为不含有转基因的可育花粉， 进一步表明 PBnOs载体的转 基因水稻植株中所有含有细胞毒性基因 BrSZl的花粉， 全部为不育花粉， 实现了载体设计 的调节花粉育性的功能。

Claims

1. 一种调控植物育性的方法， 其特征在于所述方法包括使植物含有一种构建体， 所述 构建体含有一个 Barnase基因和一个 Barstar基因。
2. 权利要求 1所述的方法，其中所述的 Barnase基因由一个花粉后期特异表达启动子驱 动表达。
3. 权利要求 2所述的方法， 其中所述的花粉后期特异表达启动子为 PG47或 Pl。
4. 权利要求 1所述的方法，其中所述的 Barstar基因由一个组成型表达启动子驱动表达。
5. 权利要求 4所述的方法， 其中所述的组成型表达启动子为 35s。
6. 一种构建体， 其特征在于所述表达盒含有一个 Barnase基因和一个 Barstar基因。
7. 权利要求 6所述的构建体，其中所述的 Barnase基因由一个花粉后期特异表达启动子 驱动表达。
8. 权利要求 7所述的构建体， 其中所述的花粉后期特异表达启动子为 PG47或 Pl。
9. 权利要求 6所述的构建体， 其中所述的 Barstar基因由一个组成型表达启动子驱动表 达。
10.权利要求 9所述的构建体， 其中所述的组成型表达启动子为 35s。
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