CN112251417A - Application of arabidopsis chlorophyll b synthetic gene CAO in tomato - Google Patents
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Abstract
The invention relates to arabidopsis chlorophyllbApplication of synthetic gene CAO in tomato for over-expressing AtCAO in tomato to change chlorophyll content and chlorophyll content of tomatoa/bThe proportion, the change of the photosynthetic system of the new germplasm created by research, the change of the chlorophyll fluorescence parameter and the photosynthesis efficiency; and analyzing the change of the agronomic characters of the new germplasm, thereby obtaining the vegetable plant with obviously improved overall photosynthesis efficiency and obviously improved yield or qualityThereby providing excellent germplasm resources which can adapt to the weak light environment for the facility gardening vegetables. And breeding a new tomato variety capable of adapting to the facility cultivation low-light environment. The implementation of the research is expected to greatly improve the yield and planting benefit of facility tomatoes in the future, meet the requirements of consumers on tomatoes in different seasons and increase the economic benefit of vegetable growers.
Description
Technical Field
The invention relates to an application of arabidopsis chlorophyll b synthetic gene CAO in tomato, belonging to the technical field of biological genetic engineering.
Background
There are 2 types of chlorophyll in higher plants: chlorophyll a and chlorophyll b, which have different absorption spectra. In the last step of chlorophyll synthesis, chlorophyll a and chlorophyll b are interconverted, known as the chlorophyll cycle. In this cycle, chlorophyll a is converted by Chlorophyll A Oxygenase (CAO) to synthesize chlorophyll b. The mutual conversion of chlorophyll a and b can strictly regulate the ratio of chlorophyll a to b: under the catalysis of CAO, the chlorophyll a synthesizes enough chlorophyll b; when an excess of chlorophyll b is produced, the ratio of chlorophyll a/b changes, causing the re-conversion of chlorophyll b to chlorophyll a. When the plants are transferred from strong light to weak light, more chlorophyll b can be gathered to a certain extent, the chlorophyll a/b ratio is reduced, the peripheral light capturing system of the photosynthesis system is increased, and the photosynthesis can be carried out by absorbing more light energy. When the plants are transferred to the strong light condition from the weak light, the chlorophyll b can be degraded to a certain extent to avoid light damage, the chlorophyll a/b ratio is improved, and the peripheral light capturing system of the photosynthetic system is reduced. However, the regulation ability of the plant itself is limited, and therefore, when the light environment in which the plant is located is changed greatly, the plant cannot completely adapt to the change of the light environment, and the photosynthesis efficiency cannot be in an optimal state.
Chlorophyll a and chlorophyll b bind to proteins of the photosynthetic system and form functional complexes with these proteins. Chlorophyll a is present in the peripheral light-harvesting complexes and the core complex of photosystems I and II, while chlorophyll b is present only in the peripheral light-harvesting complexes of photosystems. The absorption of light energy and the transmission of photosynthetic electrons need to be balanced, and the content of chlorophyll and the change of the ratio of chlorophyll a/b are very important for the adjustment of the balance. In the arabidopsis high photosynthetic efficiency gene (HPE1) deletion mutant HPE1, the total chlorophyll amount is reduced within a certain range, and the chlorophyll a/b ratio is increased, however, the plant photosynthesis efficiency is improved, which shows that optimizing the chlorophyll content and ratio is an effective strategy for changing the photosynthesis efficiency and improving the plant yield; after the BC region of the AtCAO is over-expressed in a chlorophyll a oxygenase gene AtCAO deletion mutant ch1-1, the total chlorophyll amount is increased, the chlorophyll a/b ratio is reduced to about 1.5, and the plant senescence is delayed under the short-day condition; after overexpression of intact AtCAO, the peripheral light capture complex of photosystem II (LHCII) increased, except for changes in chlorophyll content and chlorophyll a/b ratio. After the inducible expression of AtCAO in ch1-1 by the inducible expression promoter, the chlorophyll a/b ratio is gradually reduced along with the gradual synthesis of chlorophyll b, the LHCII is gradually increased, and the photosynthesis efficiency is gradually improved. The tobacco over-expressing the Arabidopsis AtCAO has the advantages that the total synthesis amount of chlorophyll b and chlorophyll is increased under the conditions of strong light or weak light, and the accumulation amount of organic substances such as starch is obviously increased. The results of these studies show that: the genetic engineering technology is utilized to change the chlorophyll content change and the chlorophyll a/b ratio, so that the photosynthesis potential of the plants is improved.
The tomatoes are one of fruits and vegetables widely cultivated in the south and the north of China, and the facility cultivated tomatoes account for a large proportion of the total production of the tomatoes. The tomato is good for light and warm, and the light saturation point is 7 multiplied by 104lx, optimum light intensity of 3 × 104-5×104lx. Insufficient illumination in the cultivation process is one of the main reasons for low yield of the facility tomatoes. In the process of facility gardening cultivation, because of shelter coverage of the greenhouse and shading of the structure of the greenhouse, the illumination is only 1/2 to 1/3 or even lower than that of external illumination; and due to season and climate reasons, the illumination time is short, so that the facility cultivated tomatoes are stressed by weak light, the plants are not strong in growth and even overgrown, the leaves are yellow and fall off, the flowering phase and the fructification phase are delayed, the yield is reduced, and the like. In addition, the early maturing cultivation period of the tomato is in low-temperature, weak-light and short-day seasons. Insufficient light is a limiting factor for cultivating strong seedlings. Therefore, it is imperative to cultivate a tomato variety special for facility horticulture, which can adapt to weak light and can efficiently absorb light energy under the weak light to carry out photosynthesis.
Disclosure of Invention
The invention aims to provide a novel tomato germplasm without influence on photosynthesis efficiency under a low-light condition so as to improve the actual yield of tomatoes, and particularly relates to application of an arabidopsis chlorophyll b synthetic gene CAO in tomatoes. The method improves the content of chlorophyll b in the leaves and fruits of the tomatoes, reduces the ratio of chlorophyll a to chlorophyll b, increases the sizes of the peripheral light-capturing antennas in the leaves and fruits of the tomatoes and finally improves the photosynthesis efficiency of the tomatoes by cloning the BC region of chlorophyll oxygenase AtCAO in the arabidopsis thaliana and transferring the cells into the tomatoes by utilizing an agrobacterium-mediated method.
The invention aims to realize the application of an arabidopsis chlorophyll b synthetic gene CAO in tomato, which is characterized by comprising the following steps:
(1) cloning a target gene sequence and constructing a plasmid; respectively cloning a BC region sequence of an AtCAO gene in arabidopsis thaliana and a gene sequence of a tag polypeptide FLAG, and connecting the BC region sequence with the FLAG to form 1 fused gene segment BC-FLAG; constructing a p35S BC-FLAG plasmid, and transgenically overexpressing BC-FLAG in tomato;
(2) detecting the expression of AtCAO in tomato; transforming tomatoes by an agrobacterium-mediated method, and screening on a corresponding selective culture medium to obtain successfully transformed plants; detecting the transcription condition of AtCAO in a transgenic strain by using PCR, and detecting the protein amount of BC-FLAG by using western-blotting and a specific antibody anti-FLAG; the successful expression and expression quantity of BC-FLAG in tomato are determined;
(3) according to the expression condition of the target gene of the obtained successfully transformed plant, selecting representative plants with different chlorophyll contents and chlorophyll a/b, breeding the subsequent generation group, and finally selecting a homozygous plant with stable heredity.
In the step (1):
cloning the sequence of the BC region of the AtCAO gene; planting arabidopsis thaliana in a laboratory artificial climate box, then cutting leaves of arabidopsis thaliana, extracting high-quality total mRNA by using an RNeasy Plant Mini Kit (QIAGEN), and then carrying out reverse transcription by using a Prime Script RT Reagent Kit (TAKANA) to obtain cDNA; designing a specific primer according to the gene sequence of the AtCAO in a TAIR database (www.arabidopsis.org), and carrying out high-fidelity amplification on the BC region of the AtCAO; the FLAG sequence was amplified with high fidelity from the vector pEarleyGate302, and then BC was bound to FLAG using overlap PCR;
constructing a p35S BC-FLAG plasmid and performing overexpression in tomato; connecting 35S with a BC-FLAG gene fragment by using overlap PCR, integrating a Fusion sequence on a pENTR vector by using In-Fusion, and integrating a target fragment into a binary expression vector pEarleyGate301 by using a GATEWAY method after determining that the sequence is correct by sequencing; agrobacterium GV3101pMP90 was then used to mediate transfer into tomato.
Planting Arabidopsis thaliana wild type Columbia (Col-0) in a laboratory artificial climate box; agrobacterium GV3101pMP90 was used to mediate transfer into "Puhong 909" tomato.
In the step (2), measuring the CAO expression quantity in the tomato transgenic line;
screening a strain with successful BC-FLAG transgenosis and planting the strain in an incubator with controllable illumination; collecting completely developed leaves of a T1 single plant, on one hand, extracting total mRNA, carrying out reverse transcription, and detecting the transcription level of a target gene by using RT-PCR; on the other hand, extracting total protein in the leaves, separating BC-FLAG protein by SDS-PAGE/western blotting, and detecting the accumulation amount of CAO by using an anti-FLAG antibody so as to determine the protein amount of BC-FLAG in each strain; selecting representative strains with different amounts of target proteins, and breeding by adopting a rapid breeding method to quickly obtain stable homozygous transgenic strains.
Detecting the chlorophyll content of a BC-FLAG over-expression strain under the condition of weak light; collecting fully developed leaves in a strain line successfully transgenic by BC-FLAG, weighing, and extracting chlorophyll by a grinding method at the liquid nitrogen temperature by using 100% of acetone stored at minus 20 ℃ in advance; detecting the contents of chlorophyll a and chlorophyll b by using an ultraviolet-visible spectrophotometer method; the chlorophyll a/b ratio is calculated from the amounts of chlorophyll a and chlorophyll b.
The method is scientific and reasonable, and by applying the arabidopsis chlorophyll b synthetic gene CAO provided by the invention to tomatoes, the target gene sequence is cloned and a plasmid is constructed. The sequence of BC region of AtCAO gene in Arabidopsis thaliana and the gene sequence of tag polypeptide FLAG are cloned respectively. The sequence of the BC region was ligated with FLAG to form 1 fused gene segment BC-FLAG. Constructing p35S BC-FLAG plasmid, and transgenically overexpressing BC-FLAG in tomato. (2) Detecting the expression of AtCAO in tomato. And transforming the tomato by using an agrobacterium-mediated method, and screening on a corresponding selective culture medium to obtain a successfully transformed plant. PCR is utilized to detect the transcription condition of AtCAO in the transgenic strain, and western-blotting and specific antibody anti-FLAG are utilized to detect the protein amount of BC-FLAG. The successful expression and the expression quantity of BC-FLAG in tomato are determined. (3) According to the expression condition of the target gene of the obtained successfully transformed plant, selecting representative plants with different chlorophyll contents and chlorophyll a/b, breeding the subsequent generation group, and finally selecting a homozygous plant with stable heredity.
Has the advantages that: the AtCAO is over-expressed in the tomato to change the chlorophyll content and chlorophyll a/b ratio of the tomato, and the change of a photosynthetic system of the created new germplasm, the change of chlorophyll fluorescence parameters and the change of photosynthesis efficiency are researched; and the change of the agronomic characters of the new germplasm is analyzed, so that a vegetable strain with obviously improved overall photosynthesis efficiency and obviously improved yield or quality is obtained, and excellent germplasm resources capable of adapting to a weak light environment are provided for the facility horticultural vegetables. And breeding a new tomato variety capable of adapting to the facility cultivation low-light environment. The implementation of the research is expected to greatly improve the yield and planting benefit of facility tomatoes in the future, meet the requirements of consumers on tomatoes in different seasons and increase the economic benefit of vegetable growers.
The tomato is one of important vegetables, the photosynthesis efficiency of the tomato is improved by improving the content of chlorophyll b, the obtained new tomato germplasm is expected to greatly improve the yield and planting benefit of facility tomatoes in the future, the requirements of consumers on the tomato in different seasons are met, and the economic benefit of vegetable growers is increased.
Drawings
FIG. 1 shows the PCR identification of the BC-F transgenic lines.
FIG. 2 shows the identification of the BC-F transgenic line western blotting.
FIG. 3 shows the chlorophyll content assay of the BC-F transgenic lines.
Detailed Description
The invention will be further illustrated with reference to specific embodiments.
(1) The sequence of the BC region of the AtCAO gene was cloned. Arabidopsis thaliana wild type Columbia (Col-0) was planted in a laboratory climatic chamber, then Arabidopsis thaliana leaves were cut, high-quality total mRNA was extracted using RNeasy Plant Mini Kit (QIAGEN), and then cDNA was obtained by reverse transcription using Prime Script RT Reagent Kit (TAKANA). Specific primers were designed based on the gene sequence of AtCAO in the TAIR database (www.arabidopsis.org) and the BC region of AtCAO was amplified with high fidelity. The FLAG sequence was amplified with high fidelity from the vector pEarleyGate302, and then BC was bound to FLAG using overlap PCR.
(2) The p35S BC-FLAG plasmid was constructed and overexpressed in tomato. 35S and BC-FLAG gene fragments are connected by utilizing overlap PCR, then a Fusion sequence is integrated on a pENTR vector by utilizing In-Fusion, after the sequence is determined to be correct by sequencing, a target fragment is integrated on a binary expression vector pEarleyGate301 by utilizing a GATEWAY method. Agrobacterium GV3101pMP90 was then used to mediate transfer into "purred 909" tomato.
(3) Measurement of the expression level of CAO in "Puhong 909" transgenic line. Screening the strain with successful BC-FLAG transgenosis and planting the strain in an incubator with controllable illumination. Collecting completely developed leaves of a T1 single plant, on one hand, extracting total mRNA, carrying out reverse transcription, and detecting the transcription level of a target gene by using RT-PCR; on the other hand, total protein in the leaf was extracted, the BC-FLAG protein was separated by SDS-PAGE/western blotting, and the amount of accumulation of CAO was detected by an anti-FLAG antibody, thereby determining the amount of BC-FLAG protein in each strain. Selecting representative strains with different amounts of target proteins, and breeding by adopting a rapid breeding method to quickly obtain stable homozygous transgenic strains.
(4) And (3) detecting the chlorophyll content of the BC-FLAG over-expression strain under the condition of weak light. Collecting fully developed leaves in a strain line successfully transgenic by BC-FLAG, weighing, and extracting chlorophyll by a grinding method at the liquid nitrogen temperature by using 100% acetone stored at minus 20 ℃ in advance. And detecting the contents of chlorophyll a and chlorophyll b by using an ultraviolet-visible spectrophotometer method. The chlorophyll a/b ratio is calculated from the amounts of chlorophyll a and chlorophyll b.
Claims (5)
1. The application of an arabidopsis chlorophyll b synthetic gene CAO in tomato is characterized by comprising the following steps:
(1) cloning a target gene sequence and constructing a plasmid; respectively cloning a BC region sequence of an AtCAO gene in arabidopsis thaliana and a gene sequence of a tag polypeptide FLAG, and connecting the BC region sequence with the FLAG to form 1 fused gene segment BC-FLAG; constructing a p35S BC-FLAG plasmid, and transgenically overexpressing BC-FLAG in tomato;
(2) detecting the expression of AtCAO in tomato; transforming tomatoes by an agrobacterium-mediated method, and screening on a corresponding selective culture medium to obtain successfully transformed plants; detecting the transcription condition of AtCAO in a transgenic strain by using PCR, and detecting the protein amount of BC-FLAG by using western-blotting and a specific antibody anti-FLAG; the successful expression and expression quantity of BC-FLAG in tomato are determined;
(3) according to the expression condition of the target gene of the obtained successfully transformed plant, selecting representative plants with different chlorophyll contents and chlorophyll a/b, breeding the subsequent generation group, and finally selecting a homozygous plant with stable heredity.
2. The use of the arabidopsis thaliana chlorophyll b synthesis gene CAO in tomato according to claim 1, wherein in step (1):
cloning the sequence of the BC region of the AtCAO gene; planting arabidopsis thaliana in a laboratory artificial climate box, then cutting leaves of arabidopsis thaliana, extracting high-quality total mRNA by using an RNeasy Plant Mini Kit (QIAGEN), and then carrying out reverse transcription by using a Prime Script RT Reagent Kit (TAKANA) to obtain cDNA; designing a specific primer according to the gene sequence of the AtCAO in a TAIR database (www.arabidopsis.org), and carrying out high-fidelity amplification on the BC region of the AtCAO; the FLAG sequence was amplified with high fidelity from the vector pEarleyGate302, and then BC was bound to FLAG using overlap PCR;
constructing a p35S BC-FLAG plasmid and performing overexpression in tomato; connecting 35S with a BC-FLAG gene fragment by using overlap PCR, integrating a Fusion sequence on a pENTR vector by using In-Fusion, and integrating a target fragment into a binary expression vector pEarleyGate301 by using a GATEWAY method after determining that the sequence is correct by sequencing; agrobacterium GV3101pMP90 was then used to mediate transfer into tomato.
3. The use of the arabidopsis thaliana chlorophyll b synthesis gene CAO in tomato, as claimed in claim 2, wherein the wild type Columbia (Col-0) of arabidopsis thaliana is planted in a laboratory climatic chamber; agrobacterium GV3101pMP90 was used to mediate transfer into "Puhong 909" tomato.
4. The use of the arabidopsis thaliana chlorophyll b synthesis gene CAO in tomato according to claim 2 or 3, wherein in step (2) of claim 1, the expression level of CAO in a transgenic line of tomato is measured;
screening a strain with successful BC-FLAG transgenosis and planting the strain in an incubator with controllable illumination; collecting completely developed leaves of a T1 single plant, on one hand, extracting total mRNA, carrying out reverse transcription, and detecting the transcription level of a target gene by using RT-PCR; on the other hand, extracting total protein in the leaves, separating BC-FLAG protein by SDS-PAGE/western blotting, and detecting the accumulation amount of CAO by using an anti-FLAG antibody so as to determine the protein amount of BC-FLAG in each strain; selecting representative strains with different amounts of target proteins, and breeding by adopting a rapid breeding method to quickly obtain stable homozygous transgenic strains.
5. The application of the arabidopsis thaliana chlorophyll b synthetic gene CAO in tomato is characterized in that the detection of the chlorophyll content of a BC-FLAG over-expression strain under the condition of low light; collecting fully developed leaves in a strain line successfully transgenic by BC-FLAG, weighing, and extracting chlorophyll by a grinding method at the liquid nitrogen temperature by using 100% of acetone stored at minus 20 ℃ in advance; detecting the contents of chlorophyll a and chlorophyll b by using an ultraviolet-visible spectrophotometer method; the chlorophyll a/b ratio is calculated from the amounts of chlorophyll a and chlorophyll b.
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2020
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Application publication date: 20210122 |