Method for creating Chinese poplars with golden leaves
阅读说明：本技术 一种金叶银中杨的创制方法 (Method for creating Chinese poplars with golden leaves ) 是由 李艺迪 姜静 刘桂丰 冮慧欣 陈肃 顾宸瑞 李慧玉 黄海娇 于 2019-10-28 设计创作，主要内容包括：本发明公开了一种金叶银中杨的PaGLK基因及应用,其核苷酸序列如SEQ ID NO：1所示。还公开金叶银中杨的创制方法,包括：克隆PaGLK基因；根据该基因保守序列设计特异性引物Ⅰ和特异性引物Ⅱ,PCR扩增分别获取靶向DNA序列和靶向DNA反向互补序列；将pFGC5941载体和靶向DNA序列分别双酶切后,连接获取pFGC5941-Cis载体；将pFGC5941-Cis载体和靶向DNA反向互补序列分别双酶切后,连接获取pGLK-RNAi载体,将该载体采用农杆介导法转化到银中杨中,获得金叶银中杨。通过构建含PaGLK基因表达载体,使PaGLK基因在转基因银中杨中低量表达,叶片呈黄色,为育种提供基础。(The invention discloses a PaGLK gene of Populus tremuloides and application thereof, wherein the nucleotide sequence of the PaGLK gene is shown as SEQ ID NO: 1 is shown. Also discloses a method for creating the populus tremuloides, which comprises the following steps: cloning the PaGLK gene; designing a specific primer I and a specific primer II according to the gene conserved sequence, and respectively obtaining a target DNA sequence and a target DNA reverse complementary sequence by PCR amplification; performing double enzyme digestion on the pFGC5941 vector and the target DNA sequence respectively, and connecting to obtain a pFGC5941-Cis vector; and performing double enzyme digestion on the pFGC5941-Cis vector and the reverse complementary sequence of the target DNA respectively, connecting to obtain a pGLK-RNAi vector, and transforming the vector into the populus tremuloides by adopting a crop-stem-mediated method to obtain the populus tremuloides. By constructing an expression vector containing the PaGLK gene, the PaGLK gene is expressed in transgenic populus argentifolia at low level, leaves are yellow, and a foundation is provided for breeding.)
1. The PaGLK gene of Populus tremuloides is characterized in that the nucleotide sequence of the PaGLK gene is shown as SEQ ID NO: 1 is shown.
2. A protein encoded by the PaGLK gene of populus tremuloides of claim 1, wherein the amino acid sequence of the protein is as set forth in SEQ ID NO: and 6.
3. An Osmunda japonica expression vector pGLK-RNAi comprising the PaGLK gene of claim 1.
4. The method for creating the populus tremuloides is characterized by comprising the following steps of:
step 1: extracting RNA of wild populus deltoides, and cloning to obtain the PaGLK gene as claimed in claim 1 by taking reverse transcribed cDNA as a template;
step 2: designing a specific primer I and a specific primer II according to the conserved sequence of the PaGLK gene, and respectively obtaining a target DNA sequence and a target DNA reverse complementary sequence through PCR amplification;
and step 3: the pFGC5941 vector and the target DNA sequence are respectively cut by NcoI and AscI and then are connected by ligase to obtain a pFGC5941-Cis vector;
the pFGC5941-Cis vector and the reverse complementary sequence of the target DNA are digested by XbaI and BamHI respectively and then are connected by ligase to obtain a pGLK-RNAi vector;
and 4, step 4: transforming the pGLK-RNAi vector into the Populus tremula pall by adopting a crop-stem-mediated method to obtain the Populus tremula pall;
wherein, the specific primer I comprises a forward primer of specific targeting DNA as shown in SEQ ID NO: 2 and the reverse primer is shown as SEQ ID NO: 3 is shown in the specification; the specific primer II comprises a forward primer of a reverse complementary sequence of the specific targeting DNA, which is shown as SEQ ID NO: 4 and the reverse primer is shown as SEQ ID NO: 5, respectively.
5. The method for creating Populus tremuloides according to claim 4, wherein the specific primer I PCR is performed in a 20. mu.L amplification system: pfu Buffer2 uL, dNTP Mix 2 uL, forward primer for specific targeting DNA 0.4 uL, reverse primer for specific targeting DNA 0.4 uL, pfu DNA polymerase0.4 u L, DNA 0.5.5 uL and the balance of sterilized water;
and (3) amplification procedure: 1min at 95 ℃; 40 cycles of 95 ℃ for 20s, 55 ℃ for 20s, 72 ℃ for 1 min; 3min at 72 ℃; keeping the temperature at 16 ℃.
6. The method for creating Populus tremuloides according to claim 4, wherein the PCR of the specific primer II is performed by using 20. mu.L of an amplification system: pfu Buffer 2. mu.L, dNTP Mix
2 μ L of forward primer specifically targeting the reverse complement sequence of DNA 0.4 μ L, pfu DNA polymerase0.4 μ L, DNA 0.5.5 μ L and the balance sterilized water;
and (3) amplification procedure: 1min at 95 ℃; 40 cycles of 95 ℃ for 20s, 55 ℃ for 20s, 72 ℃ for 1 min; 3min at 72 ℃; keeping the temperature at 16 ℃.
7. The method of creating Populus tremuloides of claim 4, wherein the pGLK-RNAi vector is transformed into Populus tremuloides in step 4 by a straw-mediated method, comprising the steps of:
s1: selecting leaves of the poplar sterile rooting seedling as a transgenic receptor, inoculating the leaves to a differentiation culture medium, and pre-culturing for 2-3 d;
s2: one day before the pre-culture is finished, inoculating the engineering bacteria containing pGLK-RNAi into LB culture solution containing antibiotics according to the volume fraction of 2%, culturing overnight at 28 ℃ and 180rpm, collecting the culture and transferring the culture into the LB culture solution without the antibiotics, and continuously culturing for 3-4 hours to obtain engineering bacteria solution;
s3: impregnating the leaves pre-cultured in the S1 with engineering bacterial liquid obtained in the S3, sucking away bacterial liquid on the leaves, inoculating the leaves on a differentiation medium, and culturing for 2 d;
s4: transferring the co-cultured leaves to a selective culture medium for selective culture, and forming resistant callus at the incision after 15 d; cutting the resistant callus and transferring the cut resistant callus to a new selective culture medium for continuous screening, and obtaining adventitious buds after 25 days; and transferring the adventitious bud into a rooting culture medium for rooting culture, and growing an adventitious root after 15 days to obtain the transgenic populus alba.
8. The method of making Populus tremuloides of claim 7, wherein the differentiation medium of S3 comprises the following components: MS + TDZ0.1mg/L + NAA 0.02mg/L + sucrose 20g/L + agar 8 g/L;
the co-culture conditions are as follows: culturing at 28 deg.C in dark.
9. The method of making Populus tremuloides of claim 7, wherein the selection medium in S4 comprises the following components: MS + TDZ0.1mg/L + NAA 0.02mg/L + sucrose 20g/L + agar 8g/L + cefuroxime 200mg/L + herbicide 1 mg/L;
the rooting medium comprises the following components: 1/4MS + IBA 0.5mg/L + sucrose 20g/L + agar 8 g/L.
10. Use of the PaGLK gene of populus tremuloides as claimed in claim 1, wherein the qualitative interference with PaGLK gene expression results in yellow leaves in the transgenic populus tremuloides.
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for creating a Populus tremuloides.
Background
Populus alba (Populus alba. P. berolinensis) is one of broadleaf tree species in northeast, but as the requirements of people on urban garden plant colors are higher and higher along with social progress and economic development, the traditional greening plant materials are single in variety and monotonous in greening landscape, and the requirements of the masses to meet the increasing social requirements are far from being met. Therefore, the research of the gene engineering breeding of populus deltoids is imperative. At present, insect-resistant, drought-resistant and salt-tolerant transgenic populus argentis has been obtained by adopting an agrobacterium-mediated method, but the color-leaf plants are just like brilliant leaves in flowers, so that the urban landscape levels are greatly enriched, the color-leaf plants become a new favorite for landscaping and beautification at present, and the color-leaf plants become the key point of research in landscape plant design in recent years. The pigments in the cells of tree leaves include chlorophyll, anthocyanin and carotenoid, and the amount and position of these pigments in the cells determine the color of the leaves. When the proportion of chlorophyll in leaf pigment is large, the leaf is green; when the anthocyanin accounts for a large proportion, the leaves are red; when the carotenoid accounts for a large proportion, the leaves appear orange or yellow. And certain parts of the leaves have high pigment content, such as the upper parts or the lower parts of the leaves, so that the colored leaf plants are formed. In a word, the creating method can enrich the composition, adjust the color, form gorgeous patterns and different seasonal results in landscape plant configuration. The method becomes a new favorite in the current urban greening and has wide development prospect.
In view of the demand of constructing color plants, no report related to the construction of color populus tremuloides by using genetic engineering is available in the prior art, so that the invention researches a creating method of the populus tremuloides and provides a basis for breeding new varieties of the populus tremuloides.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
It is still another object of the present invention to provide a PaGLK gene of Populus tremuloides which regulates leaf color.
Still another object of the present invention is to provide an expression vector pGLK-RNAi of Populus tremuloides, which carries PaGLK gene and transfers it into Populus tremuloides, and makes the PaGLK gene expressed in low amount and leaves appear yellow.
The invention also aims to provide a method for creating a new variety of the transgenic populus tremuloides, wherein a pGLK-RNAi vector is constructed, and the vector is introduced into the populus tremuloides by adopting an agrobacterium-mediated method, so that the PaGLK gene in the transgenic populus tremuloides is expressed in low quantity, leaves are yellow, and the new variety of the populus tremuloides is created.
The invention also aims to provide application of the PaGLK gene of the Populus tremula, and the Populus tremula with yellow leaves is created.
To achieve these objects and other advantages of the present invention, there is provided a PaGLK gene of populus tremuloides having a nucleotide sequence as set forth in SEQ ID NO: 1 is shown.
The invention also provides a protein encoded by the PaGLK gene of populus tremuloides as claimed in
The present invention also provides a Populus tremuloides expression vector pGLK-RNAi comprising the PaGLK gene of
The invention also provides a method for creating the populus tremuloides, which comprises the following steps:
step 1: extracting RNA of wild populus deltoides, and cloning to obtain the PaGLK gene as claimed in
and step 3: the pFGC5941 vector and the target DNA sequence are respectively cut by NcoI and AscI and then are connected by ligase to obtain a pFGC5941-Cis vector;
the pFGC5941-Cis vector and the reverse complementary sequence of the target DNA are digested by XbaI and BamHI respectively and then are connected by ligase to obtain a pGLK-RNAi vector;
and 4, step 4: transforming the pGLK-RNAi vector into the Populus tremula pall by adopting a crop-stem-mediated method to obtain the Populus tremula pall;
wherein, the specific primer I comprises a forward primer of specific targeting DNA as shown in SEQ ID NO: 2 and the reverse primer is shown as SEQ ID NO: 3 is shown in the specification; the specific primer II comprises a forward primer of a reverse complementary sequence of the specific targeting DNA, which is shown as SEQ ID NO: 4 and the reverse primer is shown as SEQ ID NO: 5, respectively.
Preferably, the specific primer I PCR is performed by using a 20 mu L amplification system: pfu Buffer2 uL,
and (3) amplification procedure: 1min at 95 ℃; 40 cycles of 95 ℃ for 20s, 55 ℃ for 20s, 72 ℃ for 1 min; 3min at 72 ℃; keeping the temperature at 16 ℃.
Preferably, the PCR of the specific primer II is performed by using a 20-L amplification system: pfu Buffer2 uL,
and (3) amplification procedure: 1min at 95 ℃; 40 cycles of 95 ℃ for 20s, 55 ℃ for 20s, 72 ℃ for 1 min; 3min at 72 ℃; keeping the temperature at 16 ℃.
Preferably, the pGLK-RNAi vector in
s1: selecting leaves of the poplar sterile rooting seedling as a transgenic receptor, inoculating the leaves to a differentiation culture medium, and pre-culturing for 2-3 d;
s2: one day before the pre-culture is finished, inoculating the engineering bacteria containing pGLK-RNAi into LB culture solution containing antibiotics according to the volume fraction of 2%, culturing overnight at 28 ℃ and 180rpm, collecting the culture and transferring the culture into the LB culture solution without the antibiotics, and continuously culturing for 3-4 hours to obtain engineering bacteria solution;
s3: impregnating the leaves pre-cultured in the S1 with engineering bacterial liquid obtained in the S3, sucking away bacterial liquid on the leaves, inoculating the leaves on a differentiation medium, and culturing for 2 d;
s4: transferring the co-cultured leaves to a selective culture medium for selective culture, and forming resistant callus at the incision after 15 d; cutting the resistant callus and transferring the cut resistant callus to a new selective culture medium for continuous screening, and obtaining adventitious buds after 25 days; and transferring the adventitious bud into a rooting culture medium for rooting culture, and obtaining the transgenic populus alba after 15 days from the adventitious root on the root.
Preferably, the differentiation medium described in S3 comprises the following components: MS + TDZ0.1mg/L + NAA 0.02mg/L + sucrose 20g/L + agar 8 g/L;
the co-culture conditions are as follows: culturing at 28 deg.C in dark.
Preferably, the selection medium in S4 comprises the following components: MS + TDZ0.1mg/L + NAA 0.02mg/L + sucrose 20g/L + agar 8g/L + cefuroxime 200mg/L +
the rooting medium comprises the following components: 1/4MS + IBA 0.5mg/L + sucrose 20g/L + agar 8 g/L.
The invention also provides application of the PaGLK gene of the Chinese populus tremuloides, which qualitatively interferes with the expression of the PaGLK gene, and the transgenic Chinese populus tremuloides presents yellow leaves.
The invention at least comprises the following beneficial effects:
the invention is based on the nucleotide sequence of the PaGLK gene of Populus tremuloides, and is obtained by constructing a plant expression vector pGLK-RNAi vector containing the PaGLK gene, wherein the vector takes a pFGC5941 vector as a basic skeleton (figure 1), a GLK targeting DNA sequence is inserted into the upstream of a CHSA intron, and a GLK targeting DNA reverse complementary sequence is inserted into the downstream of the CHSA intron. The genetic transformation of the Populus sinica leaves is carried out by utilizing an agrobacterium-mediated method, and the genetic transformation and the analysis of the leaf color characters are carried out on the gene level, and the results show that: the PaGLK gene in the transgenic populus tremuloides is expressed in low quantity, and the leaves are yellow, so that the PaGLK gene of the populus tremuloides participates in regulating and controlling the color of the leaves, and a foundation is provided for breeding new varieties of the populus tremuloides.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a map of the pFGC5941 vector of the present invention;
FIG. 2 is a schematic diagram of pGLK-RNAi interfering expression vector of the present invention;
FIG. 3 is a schematic diagram of the process for obtaining Populus alba pGLK-RNAi interference expression strain of the present invention, wherein A: selective culture after agrobacterium infection; b: transferring pGLK-RNAi poplar resistant callus; c: transferring pGLK-RNAi poplar resistant adventitious buds; d: transplanting pGLK-RNAi populus argentea; e: transplanting 3 months of pGLK-RNAi poplars;
FIG. 4 shows the Populus alba pGLK-RNAi interference expression strain of the present invention, wherein A: (left is wild type Populus alba leaf WT; right is transgenic Populus alba plant leaf); b: from left to
FIG. 5 is a PCR electrophoresis diagram of a pFGC5941-Cis Agrobacterium tumefaciens liquid of the present invention; m: DNA marker DL 2000; 1: pFGC5941-Cis plasmid; 2: water; 3-5: 3 single clones were picked;
FIG. 6 is PCR electrophoresis pattern of pGLK-RNAi Agrobacterium bacteria liquid of the present invention; m: DNA marker DL 2000; 1: pGLK-RNAi plasmid; 2: water; 3-5: 3 single clones were picked.
Detailed Description
The present invention is described in further detail below to enable those skilled in the art to practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.