WO2020147113A1 - 一种核苷酸序列及其在提高植物分泌型腺毛密度中的应用 - Google Patents

一种核苷酸序列及其在提高植物分泌型腺毛密度中的应用 Download PDF

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WO2020147113A1
WO2020147113A1 PCT/CN2019/072349 CN2019072349W WO2020147113A1 WO 2020147113 A1 WO2020147113 A1 WO 2020147113A1 CN 2019072349 W CN2019072349 W CN 2019072349W WO 2020147113 A1 WO2020147113 A1 WO 2020147113A1
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plant
target gene
nucleotide sequence
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gene
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唐克轩
谢利辉
付雪晴
秦维
黎凌
刘航
陈甜甜
钱虹妹
孙小芬
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Shanghai Jiao Tong University
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Priority to AU2020102065A priority patent/AU2020102065A4/en
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Definitions

  • the present invention relates to the technical field of plant genetic engineering, in particular to a nucleotide sequence and its application in increasing the density of plant secretory glandular hairs.
  • Glandular hair is a protruding structure derived from plant epidermal cells. According to whether the glandular hair can secrete and store secondary metabolites, it is divided into non-secretory glandular hair and secretory glandular hair. About 30% of vascular plants in nature have secretory glandular hairs on the epidermis of aerial organs, such as mint and basil in the labiatae, artemisia and sunflower in the asteraceae, tobacco and tomato in the solanaceous family, and quinoa Alfalfa, alfalfa and moraceae hops all have secretory glandular hairs.
  • Secretory glandular hairs can not only help plants resist pests and UV damage, but also secrete a lot of chemical substances, mainly terpenes, toluenes, flavonoids, methyl ketones, acyl sugars, etc., these chemicals It can be used to make medicine, produce pesticides and essential oils, and has high commercial value. Therefore, secretory glandular hairs are also called plant factories.
  • Artemisia annua also known as Artemisia annua
  • Artemisia annua is an annual traditional Chinese herbal medicine plant belonging to the genus Asteraceae. Artemisia annua has secretory glandular trichomes and T-shape non-glandular trichomes (non-glandular trichomes).
  • Artemisinin is a sesquiterpene lactone compound containing a peroxy bridge structure. Artemisinin and its derivatives are mainly used to treat malaria. Artemisinin-based combination therapy is the treatment recommended by the World Health Organization Falciparum malaria is the most effective method. Artemisinin is only synthesized and stored in the secretory glandular hairs of Artemisia annua.
  • Secretory glandular hairs are distributed in different numbers in plants. In some crops with important economic value, the insufficient number and density of secretory glandular hairs restrict the yield of its secondary metabolites.
  • the technical problem to be solved by the present invention is how to increase the density of plant secretory glandular hairs.
  • the present invention provides a nucleotide sequence selected from the following sequences:
  • SEQ ID NO: the nucleotide sequence shown in any one of 1, 3, and 5;
  • SEQ ID NO a nucleotide sequence derived from the nucleotide sequence shown in any one of 1, 3, and 5 through one or several nucleotide substitutions, deletions, or additions;
  • the present invention also provides an amino acid sequence, which is selected from the following sequences:
  • SEQ ID NO: the amino acid sequence shown in any one of 2, 4, and 6;
  • SEQ ID NO an amino acid sequence derived from the amino acid sequence shown in any one of 2, 4, and 6 through one or several amino acid substitutions, deletions, or additions;
  • the present invention also provides the application of the above-mentioned nucleotide sequence or amino acid sequence in increasing the density of plant secretory glandular hairs.
  • the above-mentioned plants are one or more of Lamiaceae plants, legumes, and Asteraceae plants.
  • the above-mentioned plants are wild peppermint (Mentha haplocalyx Briq), spearmint (Mentha spicata Linn), artichoke (Cynara cardunculus var.scolymus), sunflower (Helianthus annuus), tomato (Solanum lycopersicum), wild species of panna One or more of Solanum pennellii and Solanum tuberosum.
  • the present invention also provides a transgenic method for increasing the density of secretory glandular hairs in plants, including the following steps:
  • the gene cloning method includes the following steps: extracting total plant genome RNA, reverse transcription to synthesize cDNA, design primers according to the target gene sequence, perform PCR amplification and sequencing, to obtain the target gene.
  • step (2) constructing a plant expression vector with the target gene includes the following steps: a high-fidelity enzyme amplifies the target gene gene sequence, and introduces BamH I and Xba I restriction sites before and after the target gene, using Ligase connects the target gene with the vector, transforms the host cell, picks a single clone, and extracts the plasmid for PCR detection and restriction enzyme digestion verification.
  • step (3) the transformation of Agrobacterium tumefaciens includes the following steps: using a freeze-thaw method to transfer the plant expression vector with the target gene into Agrobacterium tumefaciens, and PCR verification.
  • the transformation includes the following steps: pre-cultivation of explants; co-cultivation of Agrobacterium and explants; screening of regenerated plants containing the target gene using a resistant medium.
  • the above-mentioned plant is Artemisia annua.
  • the above-mentioned target gene is any one of A. annua AaWRKY75b gene, wild mint MhWRKY75b gene and spearmint MsWRKY75b gene.
  • AaWRKY75b gene sequence of A. annua is shown in SEQ ID NO. 1; the wild peppermint MhWRKY75b gene sequence is shown in SEQ ID NO. 3; the spearmint MsWRKY75b gene sequence is shown in SEQ ID NO. 5.
  • amino acid sequence encoded by the AaWRKY75b gene of Artemisia annua is shown in SEQ ID NO. 2; the amino acid sequence encoded by the wild peppermint MhWRKY75b gene is shown in SEQ ID NO. 4; the amino acid sequence encoded by the spearmint MsWRKY75b gene is shown in SEQ ID Shown in NO.6.
  • step (1) the primer sequence is as shown in SEQ ID NO: 7 and 8.
  • cDNA is synthesized under the action of the reverse transcriptase PowerScript.
  • the pre-cultivation of explants includes the following steps: soak the A. annua seeds in 75% ethanol for 1 min, then soak in 20% NaClO for 20 min, rinse with sterile water 3-4 times, and absorb the surface moisture with sterile absorbent paper. Inoculate in hormone-free MS solid medium, cultivate at 25°C under 16 hours light/8 hours dark conditions, to obtain sterile seedlings of Artemisia annua. After the seedlings grow to about 5 cm, cut out sterile seedling leaf explants for use In transformation
  • the co-cultivation of Agrobacterium and explants includes the following steps: transferring the leaf explants to a co-cultivation medium supplemented with acetosyringone (AS), and dropping the activated roots of the plant expression vector containing the target gene.
  • AS acetosyringone
  • the leaf explants were fully contacted with the bacterial solution, and cultured in the dark at 28°C for 3 days.
  • the selection of resistant regenerated plants includes the following steps: the leaf explants that have been co-cultured for 3 days are transferred to a germination selection medium, which contains 6-benzylaminopurine (6-BA) , Naphthaleneacetic acid (NAA), hygromycin (Hyg) and carbenicillin (Cb); culture at 25°C, 16 hours light/8 hours dark, subculture once every two weeks, and obtain after 2-3 subcultures Hyg resistant cluster buds are cut off and transferred to a rooting medium to be cultured to root to obtain Hyg resistant regenerated Artemisia annua plants.
  • 6-BA 6-benzylaminopurine
  • NAA Naphthaleneacetic acid
  • Hyg hygromycin
  • Cb carbenicillin
  • step (6) to determine the artemisinin content in the transgenic Artemisia annua L by HPLC-ELSD, and screen to obtain transgenic artemisinin plants with increased artemisinin content.
  • the A. annua AaWRKY75b gene or a gene with 80% or more homology is overexpressed in plants with secretory glandular hairs by transgenic methods MsWRKY75b gene), which significantly increases the density of secretory glandular hairs in transgenic plants, thereby increasing the content of secondary metabolites such as artemisinin, which has extremely high practical production and application value.
  • the density of secretory glandular hairs on the leaf surface has been significantly increased in transgenic plants, thereby transforming the secretory glandular hairs of Artemisia annua into high-yield biochemical plants.
  • the HPLC-ELSD measurement also shows that the secretory glandular hairs are The content of artemisinin in plants with increased density is also significantly increased, giving full play to the plant's potential in insect resistance and the production of specific metabolites, which has extremely high practical production and application value.
  • Fig. 1 is a comparison diagram of the density of glandular hairs on the leaves of AaWRKY75b transgenic A. annua and wild-type A. annua in an embodiment of the present invention
  • Figure 2 is a graph showing the statistical results of the density of glandular hairs on the leaves of AaWRKY75b transgenic A. annua and wild-type A. annua in an embodiment of the present invention
  • Figure 3 is a diagram showing the detection results of artemisinin content of transgenic AaWRKY75b gene Artemisia annua and wild-type artemisia annua in an embodiment of the present invention
  • Figure 4 is a comparison diagram of the density of glandular hairs on the leaves of the transgenic MhWRKY75b gene or MsWRKY75b gene Artemisia annua and wild-type Artemisia annua in an embodiment of the present invention
  • FIG. 5 is a graph showing the statistical results of the density of the glandular hairs of the leaves of the transgenic MhWRKY75b gene Artemisia annua and wild-type Artemisia annua in an embodiment of the present invention
  • Fig. 6 is a graph showing the statistical results of the density of the glandular hairs of the leaves of the transgenic A. annua and wild-type A. annua in an embodiment of the present invention.
  • Agrobacterium tumefaciens EHA105 involved in the present invention has been published in "Huang Yali, Jiang Xiliang, Tian Yunlong, Guo Ping, Zhu Changxiong; Agrobacterium tumefaciens-mediated genetic transformation of Trichoderma harzianum, Chinese Journal of Biological Engineering, 2008, 28(3) ): 38-43" published in the literature.
  • Agrobacterium tumefaciens EHA105 can be obtained through publicly available commercial channels, for example, it can be purchased from CAMBIA, Australia, and the strain number is Gambar1.
  • RNA kit of TIANGEN company The leaves of Artemisia annua were quickly ground into powder in liquid nitrogen, and the total RNA of Artemisia annua was extracted using the RNA kit of TIANGEN company according to the experimental procedures of the kit. Part of the obtained RNA solution was subjected to agarose gel electrophoresis to detect the total RNA quality and spectrophotometer concentration to determine the RNA content, and the remaining part was stored at -80°C.
  • Example 1 PowerScript reverse transcriptase was used to synthesize cDNA; gene-specific primers were designed according to the sequence of the Artemisia annua AaWRKY75b gene (GenBank accession number: KX465129.1), and the primer sequence is shown in Table 1.
  • the AaWRKY75b gene of Artemisia annua was amplified from total cDNA by PCR.
  • the PCR reaction system is shown in Table 2.
  • the amplified product was recovered by gel electrophoresis and then DNA sequencing was performed.
  • the AaWRKY75b gene sequence was amplified by high-fidelity enzyme.
  • the amplified primer sequence is shown in Table 3. Among them, the BamHI restriction site was introduced into the forward primer, and the XbaI restriction site was introduced into the reverse primer.
  • Ligase was ligated to pCAMbia 1305.1 vector (purchased from Ubao Bio), and sequencing was performed to confirm the correctness of the gene.
  • the plant overexpression vector containing the AaWRKY75b gene in Example 3 was transferred into Agrobacterium tumefaciens using the freeze-thaw method, and PCR verification was performed, and the verification primers were as shown in SEQ ID NO: 11 and SEQ ID NO: 12.
  • the Agrobacterium tumefaciens strain containing the plant overexpression vector of the AaWRKY75b gene was obtained.
  • Example 5 Agrobacterium tumefaciens mediated AaWRKY75b gene transformation of Artemisia annua
  • the seeds of Artemisia annua were soaked in 75% ethanol for 1 min, and then soaked in 20% NaClO for 20 min, rinsed with sterile water for 3-4 times, dried with sterile absorbent paper, and inoculated into hormone-free MS (Murashige and Skoog, 1962) In a solid medium, culture for 5-7 days at 25°C and 16h/8h (light/dark) light to obtain sterile seedlings of Artemisia annua. After the seedling grows to about 5 cm, cut out the explants of the sterile seedling leaves for transformation.
  • the Artemisia annua explants co-cultured for 3 days were transferred to the germination screening with 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), hygromycin (Hyg), and carbenicillin (Cb) Culture medium (MS+6-BA 0.5mg/L+NAA 0.05mg/L+Hyg (overexpression) 50mg/L+Cb 500mg/L) at 25°C, 16h/8h (light/dark) light culture, each Subculture once every two weeks, after 2-3 subcultures, Hyg-resistant cluster buds can be obtained. The well-grown resistant cluster buds are cut and transferred to a rooting medium (1/2MS+Cb 125mg/L) and cultured until they take root, thereby obtaining Hyg-resistant regenerated Artemisia annua plants.
  • 6-BA 6-benzylaminopurine
  • NAA naphthaleneacetic acid
  • Hyg hygromycin
  • the plant expression vector was transformed into Agrobacterium tumefaciens to obtain an AaWRKY75b plant overexpression vector containing AaWRKY75b plant overexpression vector used to transform A. tumefaciens.
  • the constructed Agrobacterium tumefaciens strain was used to transform A. tumefaciens to obtain Transgenic Artemisia annua plants tested by PCR.
  • Transgenic Artemisia annua plants can be directly used to screen Artemisia annua plants with increased secretory glandular hair density and increased artemisinin content.
  • HPLC water alliance 2695 system is adopted, the chromatographic column is a C-18 reversed-phase silica gel column (SymmetryShieldTM C18, 5 ⁇ m, 250x 4.6mm, Waters), the mobile phase is methanol: water, and the volume ratio of methanol: water is 70:30.
  • ELSD Adopting the wateralliance 2420 system, the drift tube temperature of the evaporative light scattering detector is 40°C, the gain is 7, and the carrier gas pressure is 5bar;
  • the mobile phase is methanol: water, and when the ratio is 70%: 30%, the retention time of artemisinin is 5.1 min, and the peak shape is good.
  • the number of theoretical plates is not less than 2000 calculated by artemisinin.
  • the reference solution was injected into 2 ⁇ L, 4 ⁇ L, 6 ⁇ L, 8 ⁇ L, 10 ⁇ L under corresponding chromatographic conditions to record the spectrum and chromatographic parameters, and the peak area (Y) was used to perform regression analysis on the standard content (X, ⁇ g).
  • Y peak area
  • a total of 2 g of fresh A. annua leaves were taken from the upper, middle and lower parts of the A. annua plant, and dried in an oven at 45°C to a constant weight. Then knock the leaves from the dried branches and grind them into powder. Weigh about 0.1g of dry powder into a 2mL Eppendorf tube, add 2mL of ethanol, treat with 40W ultrasonic for 30min, centrifuge at 5000rpm for 10min, take the supernatant and filter it with 0.22 ⁇ m filter membrane, which can be used for HPLC-ELSD to determine the content of artemisinin.
  • HPLC-ELSD was used to determine the artemisinin content
  • the sample injection volume was 20 ⁇ L
  • the artemisinin content (mg) in the sample was calculated according to the peak area into the linear regression equation, and then divided by the dry weight of the artemisia annua leaf (g) , So as to calculate the content of artemisinin in A. annua plants.
  • the results are shown in Figure 2.
  • the artemisinin content of AaWRKY75b transgenic Artemisia annua plants was significantly correlated with glandular hair density. With the increase of secretory glandular hair density, the artemisinin content increased to 18.8mg/g, wild type It is 10.5mg/g, which is about 1.8 times that of the wild type.
  • the originality is 89%; the sequence homology with the sunflower (Helianthus annuus) probable WRKY transcription factor 75 is 86%; the sequence homology with the tomato (Solanum lycopersicum) WRKY transcription factor 75 is 86%; and the wild species Pannali tomato
  • the sequence homology of (Solanum pennellii)probable WRKY transcription factor 75 is 86%; the sequence homology with the potato (Solanum tuberosum) WRKY1 gene is 85%; with the spearmint (Mentha spicata Linn) homologous gene MsWRKY75b (GenBank) Accession number: KT372786.1)
  • the sequence homology is 80%; the sequence homology with the wild peppermint (Mentha haplocalyx Briq) homologous gene MhWRKY75b (shown in SEQ ID NO: 3) is 80%.
  • Example 2 The specific operation is carried out according to the steps in Example 2, wherein the primers used are shown in Table 4, the obtained full-length coding sequence of the spearmint MsWRKY75b gene is shown in SEQ ID NO: 3, and the protein coding sequence is deduced as SEQ ID NO: 4, the full-length coding sequence of the wild peppermint MhWRKY75b gene is obtained as shown in SEQ ID NO: 5, and its protein coding sequence is deduced as shown in SEQ ID NO: 6.
  • the specific operation was performed according to the steps in Example 3, and the primer sequences used are shown in Table 5.
  • the obtained plant overexpression vectors are MhWRKY75b-pCAMbia 1305.1 vector and MsWRKY75b-pCAMbia 1305.1 vector.
  • the specific operation is carried out according to the steps in Example 4.
  • the verification primers are shown in SEQ ID NO: 11 and SEQ ID NO: 18.
  • Example 7 The specific operation was carried out according to the steps in Example 7. The results are shown in Figure 4, Figure 5 and Figure 6, where CK represents the wild-type control group.
  • the density of glandular hairs on the leaves of the transgenic A. annua plants with MsWRKY75b gene is significantly higher than that of the wild-type A. annua. up to 38.65 / mm 2, the wild type was 20.34 / mm 2 to reach 1.9 times the wild-type A. annua; MhWRKY75b transfected gene in leaves of Artemisia annua trichome density significantly higher than the wild type A. annua, up to 36.45 / mm 2 , 20.10/mm 2 , which is about 1.8 times of wild-type Artemisia annua.

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Abstract

提供一种核苷酸序列及其在提高植物分泌型腺毛密度中的应用，涉及植物基因工程技术领域，所述核苷酸序列选自如下序列：1)SEQ ID NO：1、3、5中任一项所示的核苷酸序列；2)SEQ ID NO：1、3、5中任一项所示的核苷酸序列通过一个或儿个核苷酸的取代、缺失、或添加衍生产生的核苷酸序列；3)与SEQ ID NO：1、3、5中的任一项具有至少80％同源性的核苷酸序列。通过基因工程手段将上述核苷酸序列中的任意一种转入植物体中，能够显著提高植物分泌型腺毛密度，充分发挥其在抗虫以及生产特定代谢物质上的潜力，具有极高的实际生产应用价值。

Description

一种核苷酸序列及其在提高植物分泌型腺毛密度中的应用 技术领域
本发明涉及植物基因工程技术领域，尤其涉及一种核苷酸序列及其在提高植物分泌型腺毛密度中的应用。
背景技术
腺毛是衍生于植物表皮细胞的突起结构，根据腺毛是否能分泌和储存次生代谢产物将其分为非分泌型腺毛和分泌型腺毛。自然界约30％的维管植物的气生器官表皮上都分布有分泌型腺毛，例如唇形科植物薄荷和罗勒，菊科植物青蒿和向日葵，茄科植物烟草和番茄，豆科植物藜苜蓿和紫花苜蓿以及桑科植物啤酒花等都具有分泌型腺毛。分泌型腺毛不仅能帮助植物抵抗病虫害和紫外线伤害，还能分泌大量的化学物质，主要有萜类，类甲基苯烷类，黄酮类，甲基酮类，酰基糖类等，这些化学物质可以用来制药，生产农药以及精油等，具有很高的商业价值。所以分泌型腺毛也被称为植物工厂。这其中，青蒿(Artemisia annua)又名黄花蒿，是菊科蒿属一年生的传统中草药植物，青蒿具有分泌型腺毛(glandular trichomes)和T-shape非分泌型腺毛(non-glandular trichomes)；青蒿素是一种含有过氧桥结构的倍半萜内酯化合物，青蒿素及其衍生物主要用于治疗疟疾，以青蒿素为基础的联合疗法是世界卫生组织推荐的治疗恶性疟疾最为有效的方法。而青蒿素只在青蒿的分泌型腺毛中合成和储存。
分泌型腺毛在植株中分布数量不一，在一些具有重要经济价值的作物中，分泌型腺毛的数量、密度的不足制约了其次生代谢产物的产量。
因此，本领域的技术人员致力于开发一种通过转基因技术手段提高植物分泌型腺毛密度的方法。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是如何提高植物分泌型腺毛密度。
为实现上述目的，本发明提供了一种核苷酸序列，所述核苷酸序列选自如下序列：
1)SEQ ID NO：1、3、5中任一项所示的核苷酸序列；
2)SEQ ID NO：1、3、5中任一项所示的核苷酸序列通过一个或几个核苷酸的取代、缺失、或添加衍生产生的核苷酸序列；
3)与SEQ ID NO：1、3、5中的任一项具有至少80％同源性的核苷酸序列。
另一方面，本发明还提供了一种氨基酸序列，所述氨基酸序列选自如下序列：
1)SEQ ID NO：2、4、6中任一项所示的氨基酸序列；
2)SEQ ID NO：2、4、6中任一项所示的氨基酸序列通过一个或几个氨基酸的取代、缺失、或添加衍生产生的氨基酸序列；
3)与SEQ ID NO：2、4、6中的任一项具有至少80％同源性的氨基酸序列。
另一方面，本发明还提供了上述核苷酸序列或氨基酸序列在提高植物分泌型腺毛密度中的应用。
进一步地，上述植物为唇形科植物、豆科植物、菊科植物中一种或几种。
进一步地，上述植物为野薄荷(Mentha haplocalyx Briq)、留兰香薄荷(Mentha spicata Linn)、朝鲜蓟(Cynara cardunculus var.scolymus)、向日葵(Helianthus annuus)、番茄(Solanum lycopersicum)、野生种潘那利番茄(Solanum pennellii)和马铃薯(Solanum tuberosum)中的一种或多种。
另一方面，在一个具体实施方式中，本发明还提供了一种转基因提高植物分泌型腺毛密度的方法，包括以下步骤：
(1)采用基因克隆方法获得目的基因，所述目的基因为上述核苷酸序列中的任意一种；
(2)构建具有所述目的基因的植物表达载体；
(3)将所述具有目的基因的植物表达载体转化根癌农杆菌，获得含目的基因植物表达载体的根癌农杆菌菌株；
(4)将含目的基因植物表达载体的根癌农杆菌菌株转化植物，PCR检测获得整合了所述目的基因的转基因植物植株；
(5)统计整合了目的基因的转基因植物叶片上的腺毛密度，获得腺毛密度提高的植物植株。
进一步地，在步骤(1)中，基因克隆方法包括以下步骤：提取植物基因组总RNA，反转录合成cDNA，根据目的基因序列设计引物进行PCR扩增并测序，获得目的基因。
进一步地，在步骤(2)中，构建具有目的基因的植物表达载体包括以下步骤：高保真酶扩增目的基因基因序列，并在目的基因前后分别引入BamH I和Xba I酶切位点，利用连接酶将目的基因与载体连接，转化宿主细胞，挑取单克隆，提质粒做PCR检测和酶切验证。
进一步地，在步骤(3)中，转化根癌农杆菌包括以下步骤：采用冻融法将具有目的基因的植物表达载体转入根癌农杆菌，PCR验证。
进一步地，在所骤(4)中，转化包括以下步骤：外植体的预培养；农杆菌与外植体的共培养；利用抗性培养基筛选含有目的基因的再生植株。
进一步地，在一个具体实施方式中，上述植物为青蒿。
进一步地，上述目的基因为青蒿AaWRKY75b基因、野薄荷MhWRKY75b基因和留兰香薄荷MsWRKY75b基因中的任意一种。
进一步地，青蒿AaWRKY75b基因序列如SEQ ID NO.1所示；野薄荷MhWRKY75b 基因序列如SEQ ID NO.3所示；留兰香薄荷MsWRKY75b基因如序列如SEQ ID NO.5所示。
进一步地，青蒿AaWRKY75b基因编码的氨基酸序列如SEQ ID NO.2所示；野薄荷MhWRKY75b基因编码的氨基酸序列如SEQ ID NO.4所示；留兰香薄荷MsWRKY75b基因编码的氨基酸序列如SEQ ID NO.6所示。
进一步地，在步骤(1)中，引物序列如SEQ ID NO：7和8所示。
进一步地，cDNA在反转录酶PowerScript的作用下合成。
进一步地，外植体的预培养包括以下步骤：青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS固体培养基中，25℃、16小时光照/8小时黑暗条件下培养，获得青蒿无菌苗，待苗长至5cm左右后，剪取无菌苗叶片外植体用于转化；
进一步地，农杆菌与外植体的共培养包括以下步骤：将叶片外植体转到添加乙酰丁香酮(AS)的共培养培养基中，滴加活化好的含目的基因植物表达载体的根癌农杆菌的1/2MS菌液，使所述叶片外植体与所述菌液充分接触，28℃暗培养3天。
进一步地，抗性再生植株的筛选包括以下步骤：将共培养3天的叶片外植体转入到的发芽筛选培养基中，所述发芽筛选培养基含有6-苄氨基嘌呤(6-BA)、萘乙酸(NAA)、潮霉素(Hyg)和羧苄青霉素(Cb)；25℃、16小时光照/8小时黑暗条件下培养，每两周继代培养一次，经过2-3次继代后获得Hyg抗性丛生芽，将生长良好的抗性丛生芽剪下转入生根培养基上培养至生根，获得Hyg抗性再生青蒿植株。
进一步地，还包括步骤(6)对转基因青蒿中青蒿素含量进行HPLC-ELSD测定，筛选获得青蒿素含量提高的转基因青蒿植株。
本发明的具体实施方式中，通过转基因手段在具有分泌型腺毛的植物中过量表达青蒿AaWRKY75b基因或与之具有80％及以上同源性的基因(比如野薄荷MhWRKY75b基因或留兰香薄荷MsWRKY75b基因)，显著提高了转基因植株中分泌型腺毛的密度，从而提高了其中次生代谢产物如青蒿素的含量，具有极高的实际生产应用价值。对于植物青蒿，获得叶片表面分泌型腺毛的密度得到显著提高转基因植株，从而将青蒿的分泌型腺毛转化为高产量的生化工厂，而通过HPLC-ELSD测定也表明分泌型腺毛的密度提高的植株青蒿素含量也显著提高，充分发挥植物在抗虫以及生产特定代谢物质上的潜力，具有极高的实际生产应用价值。
以下将结合附图对本发明的构思、具体实施步骤以及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个实施例中转AaWRKY75b基因青蒿和野生型青蒿叶片腺毛密度对比图；
图2是本发明的一个实施例中转AaWRKY75b基因青蒿和野生型青蒿叶片腺毛密度统计结果图；
图3是本发明的一个实施例中转AaWRKY75b基因青蒿和野生型青蒿青蒿素含量检测结果图；
图4是本发明的一个实施例中转MhWRKY75b基因或MsWRKY75b基因青蒿和野生型青蒿叶片腺毛密度对比图；
图5是本发明的一个实施例中转MhWRKY75b基因青蒿和野生型青蒿叶片腺毛密度统计结果图；
图6是本发明的一个实施例中转MsWRKY75b基因青蒿和野生型青蒿叶片腺毛密度统计结果图。
具体实施方式
下面结合附图和实施例对本发明的技术内容做进一步的说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆：实验室手册见New York：Cold Spring Harbor Laboratory Press的1989年版中所述的条件，或按照制造厂商所建议的条件。
本发明涉及的根癌农杆菌EHA105已在《黄亚丽，蒋细良，田云龙，郭萍，朱昌雄；根癌农杆菌介导的哈茨木霉菌遗传转化的研究，中国生物工程杂志，2008，28(3)：38-43》文献中公开。根癌农杆菌EHA105可通过公开市售的商业渠道取得，如可以从澳大利亚CAMBIA公司购得，菌株编号为Gambar1。
实施例1
青蒿总RNA提取
取青蒿叶片在液氮中迅速研磨成粉末，使用TIANGEN公司的RNA试剂盒，按照该试剂盒的实验步骤说明提取青蒿总RNA。得到的RNA溶液取部分进行琼脂糖凝胶电泳检测鉴定总RNA质量及分光光度计浓度测定RNA含量，剩余部分-80℃保存。
实施例2
青蒿AaWRKY75b基因的克隆
以实施例1中提取的总RNA为模板，利用PowerScript反转录酶合成cDNA；根据青蒿AaWRKY75b基因(GenBank登录号：KX465129.1)的序列设计基因特异性引物，引物序列如表1所示，通过PCR从总cDNA中扩增得到青蒿的AaWRKY75b基因，PCR反应体系如表2所示，扩增产物凝胶电泳回收后进行DNA测序。测序后获得该基因的全长编码序列，如SEQ ID NO：1所示，其中，起始密码子为ATG，终止密码子为TAG，并推导出其蛋白编码序列，如SEQ ID NO：2所示。
表1 AaWRKY75b基因克隆PCR引物
Figure PCTCN2019072349-appb-000001
表2 PCR的反应体系
Figure PCTCN2019072349-appb-000002
实施例3
含AaWRKY75b基因的植物过表达载体的构建
通过高保真酶扩增AaWRKY75b基因序列，扩增引物序列如表3所示，其中，正向引物中引入了BamHI的酶切位点，反向引物中引入了XbaI的酶切位点，再通过连接酶连接到pCAMbia 1305.1载体(购自优宝生物)上，进行测序确认基因的正确性。
表3 AaWRKY75b-pCAMbia 1305.1载体构建的PCR引物
Figure PCTCN2019072349-appb-000003
实施例4
含AaWRKY75b过量表达的根癌农杆菌工程菌的获得
将实施例3中含AaWRKY75b基因的植物过表达载体采用冻融法转入根癌农杆菌，并进行PCR验证，验证引物如SEQ ID NO：11和SEQ ID NO：12所示。获得含AaWRKY75b基因的植物过表达载体的根癌农杆菌菌株。
实施例5：根癌农杆菌介导AaWRKY75b基因转化青蒿
(1)外植体的预培养
青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS(Murashige and Skoog，1962)固体培养基中，25℃、16h/8h(光照/黑暗)光照培养5-7天，即可获得青蒿无菌苗。待苗长至5cm左右后，剪取无菌苗叶片外植体用于转化。
(2)农杆菌与外植体的共培养
将所述叶片外植体，转到添加乙酰丁香酮(AS)的共培养培养基(1/2MS+AS 100μmol/L)中，滴加含活化好的所述含AaWRKY75b植物过量表达载体的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3天。以滴加不带有目的基因的根癌农杆菌的1/2MS液体培养基悬液的叶片外植体为对照。
(3)抗性再生植株的筛选
将所述共培养3天的青蒿外植体转入到添加6-苄氨基嘌呤(6-BA)、萘乙酸(NAA)、潮霉素(Hyg)、羧苄青霉素(Cb)的发芽筛选培养基(MS+6-BA 0.5mg/L+NAA 0.05mg/L+Hyg(过表达)50mg/L+Cb 500mg/L)上于25℃、16h/8h(光照/黑暗)光照培养，每两周继代培养一次，经过2-3次继代后即可获得Hyg抗性丛生芽。将生长良好的抗性丛生芽剪下转入生根培养基(1/2MS+Cb 125mg/L)上培养至生根，从而获得Hyg抗性再生青蒿植株。
实施例6
转基因青蒿植株的PCR检测
根据目的基因所在表达盒上游的35S启动子区域和AaWRKY75b分别设计正向引物(35SF：GAAGATGCCTCTGCCGACAGTG；SEQ ID NO：11)和反向引物(AaWRKY75b-RP：GCTCTAGACTAAAACAAAGGTGGATCTTGTA；SEQ ID NO：12)对目的基因进行检测。结果表明，利用所设计的PCR特异引物，能扩增出特异DNA片段。而以非转化青蒿基因组DNA为模板时，没有扩增出任何片段。
本实施例将所述的植物表达载体转化根癌农杆菌，获得用于转化青蒿的含AaWRKY75b植物过量表达载体的根癌农杆菌菌株，利用所构建的根癌农杆菌菌株转化青蒿，获得经PCR检测的转基因青蒿植株。转基因青蒿植株可直接用于筛选分泌型腺毛密度增加并且青蒿素含量升高的青蒿植株。
实施例7：
转基因青蒿表皮腺毛密度和总腺毛数量的统计
使用奥林巴斯公司的BX51型号显微镜，在波长为450nm-480nm的激发光下观察非转基因青蒿和AaWRKY75b过量表达转基因青蒿的叶片。取同等大小的青蒿叶片，在不同的5个位置随机取样，利用ImageJ软件测量青蒿叶片总面积，统计腺毛密度，结果如图1和图2所示，其中CK表示野生型对照组，转AaWRKY75b基因青蒿植株叶片腺毛密度显著高于野生型青蒿，最高为41.59个/mm 2，野生型青蒿为20.65，约为野生型青蒿的2倍。
实施例8
利用HPLC-ELSD测定转基因青蒿中青蒿素含量
(1)HPLC-ELSD条件及系统适用性以及标准溶液的配制
HPLC：采用water alliance 2695系统，色谱柱为C-18反相硅胶柱(SymmetryShield TM C18，5μm，250x 4.6mm，Waters)，流动相为甲醇∶水，甲醇∶水的体积比为70∶30，柱温30℃，流速1.0mL/min，进样量10μL，灵敏度(AUFS＝1.0)，理论塔板数按青蒿素峰计算不低于2000。
ELSD：采用water alliance 2420系统，蒸发光散射检测器漂移管温度40℃，放大系数(gain)为7，载气压力5bar；
精密称取青蒿素标准品(Sigma公司)2.0mg用1mL甲醇完全溶解，得到2mg/mL青蒿素标准品溶液，保存于-20℃备用。
本发明中流动相为甲醇(methanol)：水，比例为70％：30％时，青蒿素的保留时间为5.1min，峰型良好。理论塔板数按青蒿素计算不低于2000。
(2)标准曲线的制作
将所述对照品溶液在相应色谱条件下分别进样2μL，4μL，6μL，8μL，10μL记录图谱及色谱参数，分别以峰面积(Y)对标准品含量(X，μg)进行回归分析。通过研究，本实施例中青蒿素在4-20g范围内呈现良好的log-log线性关系。青蒿素对照品的log-log线性回归方程为：Y＝1.28e+000X+4.71e+000，R 2＝0.979546。
(3)样品的制备和青蒿素含量的测定
在青蒿植株的上、中和下部共取2g新鲜的青蒿叶片，于45℃烘箱中烘至恒重。然后从烘干的枝条上敲下叶片磨成粉末。称取约0.1g干粉于2mL Eppendorf管中，加入2mL乙醇，用40W超声波处理30min，5000rpm离心10min，取上清用0.22μm滤膜过滤，即可用于HPLC-ELSD测定青蒿素的含量。
采用HPLC-ELSD测定青蒿素含量，样品进样体积为20μL，根据峰面积代入线形回归方程计算出样品中的青蒿素含量(mg)，再除以样品的青蒿叶干重(g)，从而计算出青蒿植株中青蒿素的含量。结果如图2所示，转AaWRKY75b基因青蒿植株的青蒿素含量与腺毛密度呈明显相关性，随着分泌型腺毛密度的提高，青蒿素含量提高至18.8mg/g，野生型为10.5mg/g，约为野生型的1.8倍。
实施例9
青蒿AaWRKY75b基因序列同源性比对
利用NCBI数据库，将如SEQ ID NO：1所示的AaWRKY75b基因序列进行核酸序列同源性比对，结果显示，该段序列与朝鲜蓟(Cynara cardunculus var.scolymus)probable WRKY transcription factor 75的序列同源性为89％；与向日葵(Helianthus annuus)probable WRKY transcription factor 75的序列同源性为86％；与番茄(Solanum lycopersicum)WRKY transcription factor 75序列同源性86％；与野生种潘那利番茄(Solanum pennellii)probable WRKY transcription factor 75的序列同源性为86％；与马铃薯(Solanum tuberosum)WRKY1 gene序列同源性为85％；与留兰香薄荷(Mentha spicata Linn)同源基因MsWRKY75b(GenBank登录号：KT372786.1)序列同源性为80％；与野薄荷(Mentha haplocalyx Briq)同源基因MhWRKY75b(如SEQ ID NO：3所示)序列同源性为80％。
实施例10
留兰香薄荷和野薄荷总RNA的提取
具体操作按照实施例1中的步骤进行。
实施例11
野薄荷MhWRKY75b基因和留兰香薄荷MsWRKY75b基因的克隆
具体操作按照实施例2中的步骤进行，其中所用引物如表4示，获得的留兰香薄荷MsWRKY75b基因的全长编码序列如SEQ ID NO：3所示，并推导出其蛋白编码序列如SEQ ID NO：4所示，获得了野薄荷MhWRKY75b基因的全长编码序列如SEQ ID NO：5所示，并推导出其蛋白编码序列如SEQ ID NO：6所示。
表4 MhWRKY75b基因和MsWRKY75b基因克隆PCR引物
Figure PCTCN2019072349-appb-000004
实施例12
含MsWRKY75b基因或MhWRKY75b基因的植物过表达载体的构建
具体操作按照实施例3中的步骤进行，所用引物序列如表5所示。获得的植物过量表达载体为MhWRKY75b-pCAMbia 1305.1载体和MsWRKY75b-pCAMbia 1305.1载体。
表5 MhWRKY75b-pCAMbia 1305.1载体和MsWRKY75b-pCAMbia 1305.1载体构建PCR引物
Figure PCTCN2019072349-appb-000005
实施例13
含MhWRKY75b基因或MsWRKY75b基因过量表达的根癌农杆菌工程菌的获得
具体操作按照实施例4中的步骤进行。验证引物如SEQ ID NO：11和SEQ ID NO：18所示。
实施例14
根癌农杆菌介导MsWRKY75b基因或MhWRKY75b基因转化青蒿
具体操作按照实施例5中的步骤进行。
实施例15
转MsWRKY75b基因或MhWRKY75b基因青蒿植株的PCR检测
具体操作按照实施例6中的步骤进行。其中用于鉴定的引物如SEQ ID NO：11和SEQ ID NO：18所示。
实施例16
转MsWRKY75b基因或MhWRKY75b基因青蒿表皮腺毛密度和总腺毛数量的统计
具体操作按照实施例7中的步骤进行，结果如图4、图5及图6所示，其中CK表示野生型对照组，转MsWRKY75b基因青蒿植株叶片腺毛密度明显高于野生型青蒿，最高为38.65个/mm 2，野生型为20.34个/mm 2达到野生型青蒿的1.9倍；转MhWRKY75b基因青蒿植株叶片腺毛密度显著高于野生型青蒿，最高为36.45个/mm 2，20.10个/mm 2，约为野生型青蒿的1.8倍。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创 造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
Figure PCTCN2019072349-appb-000006
Figure PCTCN2019072349-appb-000007
Figure PCTCN2019072349-appb-000008
Figure PCTCN2019072349-appb-000009
Figure PCTCN2019072349-appb-000010
Figure PCTCN2019072349-appb-000011
Figure PCTCN2019072349-appb-000012
Figure PCTCN2019072349-appb-000013
Figure PCTCN2019072349-appb-000014

Claims (10)

  1. 一种核苷酸序列，其特征在于，所述核苷酸序列选自如下序列：
    1)SEQ ID NO：1、3、5中任一项所示的核苷酸序列；
    2)SEQ ID NO：1、3、5中任一项所示的核苷酸序列通过一个或几个核苷酸的取代、缺失、或添加衍生产生的核苷酸序列；
    3)与SEQ ID NO：1、3、5中的任一项具有至少80％同源性的核苷酸序列。
  2. 一种氨基酸序列，其特征在于，所述氨基酸序列选自如下序列：
    1)SEQ ID NO：2、4、6中任一项所示的氨基酸序列；
    2)SEQ ID NO：2、4、6中任一项所示的氨基酸序列通过一个或几个氨基酸的取代、缺失、或添加衍生产生的氨基酸序列；
    3)与SEQ ID NO：2、4、6中的任一项具有至少80％同源性的氨基酸序列。
  3. 如权利要求1所述的核苷酸序列或如权利要求2所述的氨基酸序列在提高植物分泌型腺毛密度中的应用。
  4. 如权利要求3所述的应用，其特征在于，所述植物为野薄荷(Mentha haplocalyx Briq)、留兰香薄荷(Mentha spicata Linn)、朝鲜蓟(Cynara cardunculus var.scolymus)、向日葵(Helianthus annuus)、番茄(Solanum lycopersicum)、野生种潘那利番茄(Solanum pennellii)和马铃薯(Solanum tuberosum)中的一种或多种。
  5. 一种转基因提高植物分泌型腺毛密度的方法，其特征在于，包括以下步骤：
    (1)采用基因克隆方法获得目的基因，所述目的基因为权利要求1所述的核苷酸序列中的任意一种；
    (2)构建具有所述目的基因的植物表达载体；
    (3)将所述具有目的基因的植物表达载体转化根癌农杆菌，获得含目的基因植物表达载体的根癌农杆菌菌株；
    (4)将所述含目的基因植物表达载体的根癌农杆菌菌株转化植物，PCR检测获得整合了所述目的基因的转基因植物植株；
    (5)统计整合了目的基因的转基因植物叶片上的腺毛密度，获得腺毛密度提高的植物植株。
  6. 如权利要求5所述的转基因提高植物分泌型腺毛密度的方法，其特征在于，在步骤(1)中，所述基因克隆方法包括以下步骤：提取植物基因组总RNA，反转录合成cDNA，以SEQ ID NO：7和8所示的核苷酸序列为引物进行PCR扩增并测序，获得目的基因。
  7. 如权利要求5所述的转基因提高植物分泌型腺毛密度的方法，其特征在于，在步骤(2)中，所述构建具有目的基因的植物表达载体包括以下步骤：高保真酶扩增目的基因基因序列，并在目的基因前后分别引入BamH I和Xba I酶切位点，利用连接 酶将目的基因与载体连接，转化宿主细胞，挑取单克隆，提质粒做PCR检测和酶切验证。
  8. 如权利要求5所述的转基因提高植物分泌型腺毛密度的方法，其特征在于，在步骤(3)中，所述转化根癌农杆菌包括以下步骤：采用冻融法将具有目的基因的植物表达载体转入根癌农杆菌，PCR验证。
  9. 如权利要求5所述的转基因提高植物分泌型腺毛密度的方法，其特征在于，在步骤(4)中，所述转化包括以下步骤：外植体的预培养；农杆菌与外植体的共培养；利用抗性培养基筛选含有目的基因的再生植株。
  10. 如权利要求5所述的转基因提高植物分泌型腺毛密度的方法，其特征在于，所述植物为青蒿，所述方法还包括步骤(6)对转基因青蒿中青蒿素含量进行HPLC-ELSD测定，筛选获得青蒿素含量提高的转基因青蒿植株。
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