CN109504687A - A kind of soybean lipid transport protein gene GmABCA9 and its application - Google Patents

Abstract

The invention discloses a kind of soybean lipid transport protein gene GmABCA9 and its applications, the present invention specifies the characteristic of specific expressed and endoplasmic reticulum positioning of the soybean GmABCA9 gene in soya seeds, the development of the expression regulation arabidopsis seed of the gene, influence seed fatty acid content, show that the gene participates in the movement system of endoplasmic reticulum lipid, and then regulates and controls the synthesis of seed grease.

Description

A kind of soybean lipid transport protein gene GmABCA9 and its application

Technical field

The invention belongs to genetic engineering fields, and in particular to a kind of soybean lipid transport protein gene and its application.

Background technique

Soybean is the main source of the important oil crops in China and mankind's high-quality protein.It is both China chief crop One of and the maximum agricultural product of China's import volume.In recent years, the oil production of domestic soybean crops, which is no longer satisfied, increasingly increases The vegetable oil demand added, soya-bean oil is based on imported oil on domestic market.Therefore, domestic high-oil-content soybean varieties are cultivated, are improved State's producing soybean oil yield is extremely urgent.

To cultivate high yield high-oil-content soybean varieties, researcher regulates and controls the rouge in fat metabolic approach by genetic engineering means Fat acid enzyme and key transcription factor, but 10%~20% or so is only maintained to the promotion of fat content.

The synthesis of grease carries out in chloroplaset, transports endoplasmic reticulum later and is processed.Currently, green in leaf about grease The research of transportation system is less between body and endoplasmic reticulum, and there is not been reported in soybean.

Summary of the invention

The technical problems to be solved by the invention are as follows: how further to improve soybean grease content.

The technical solution of the present invention is as follows: soybean GmABCA9 gene, nucleotide sequence is as shown in SEQ ID No.1.

Application of the soybean GmABCA9 gene as shown in SEQ ID No.1 in regulation vegetable seeds grease content.

Further, the plant is leguminous plant.

Further, the plant is soybean.

Further, the plant is arabidopsis.

Expression vector containing the soybean GmABCA9 gene as shown in SEQ ID No.1 is in plant seed lipid regulation Application.

The protein of the coding of soybean GmABCA9 gene shown in SEQ ID No.1, amino acid sequence such as SEQ ID Shown in No.2.

GmABCA9 can be expressed in plant leaf, stem, root, seed.It, should in the seed development later period of soybean mature The expression quantity of gene is higher.

GmABCA9 is in tobacco leaf after transient expression, using microscope to the tobacco leaf and protoplast after conversion Fluorescence signal observation is carried out, discovery GmABCA9 is located in endoplasmic reticulum.

Arabidopsis abca9 mutant seeds are shrivelled, and grain is small.When GmABCA9 overexpression in arabidopsis, back mutation Body seed has restored the phenotype as wild type.When growing in no sucrose culture medium, the growth of abca9 mutant seedlings is slow Slowly, depauperation, when GmABCA9 overexpression in arabidopsis, revertant seedling development is normal.These the result shows that The Seed development of GmABCA9 regulation transgenic arabidopsis.

Overexpression GmABCA9 improves seed grease and C16:0, C18:1, C18:2 and C18:3 content of fatty acid.Table Bright GmABCA9 promotes the accumulation of grease by regulation seed fatty acid content.

Compared with prior art, the invention has the following advantages:

The present invention specifies the spy of specific expressed and endoplasmic reticulum positioning of the soybean GmABCA9 gene in soya seeds Property, the development of the expression regulation arabidopsis seed of the gene influences seed fatty acid content, shows that the gene participates in endoplasmic reticulum rouge The movement system of matter, and then regulate and control the synthesis of seed grease.

Detailed description of the invention

Fig. 1: the GmABCA9 expression in soybean different tissues；Leaves: blade；Roots: root；Seeds: kind Son；Pods: fruit pod；Stems: stem；Blade, root and stem are the tissue for sowing the soybean seedling plant of 30d, and seed and fruit pod are to open The soyabean tissue of rear 30d is spent, reference gene is soybean ACTIN.

Fig. 2: 35S:GmABCA9-GFP vector construction；A in figure, vector construction schematic diagram.B in figure, 1,2 respectively represent sample Product 1 and sample 2；M5:Marker 5000.35S:GmABCA9-GFP segment for occurring after BamH I digestion: 1523bp and 840bp。

Fig. 3: GmABCA9 in tobacco leaf fluorescence signal；A, B, C, D are tobacco leaf in figure；E, F, G, H are cigarette in figure The protoplast of blade of grass piece.

Fig. 4: 35S:GmABCA9-GFP transgenic Arabidopsis plants PCR identification；M5: molecular labeling Marker 5000；-: With dH₂O is the negative control of template；WT: wild-type Arabidopsis plants；1~15: transgenic Arabidopsis plants.

Fig. 5: the GmABCA9 expression in arabidopsis different tissues；Roots: root；Siliques: silique； Flowers: flower；Stems: stem；Rosette Leaves: lotus throne leaf；Cauline Leaves: stem leaf.Silique is sowing 45d Arabidopsis plant tissue, root, flower, stem, lotus throne leaf, stem leaf be sow 30d arabidopsis, reference gene is arabidopsis ACTIN2。

Fig. 6: the screening of arabidopsis abca9 revertant plant；M5: molecular labeling Marker 5000；- 1: with dH₂O For the negative control of template；- 2: using abca9 mutant gene group DNA as the negative control of template；WT: wildtype Arabidopsis thaliana is planted Strain；1~14: arabidopsis abca9 revertant plant.

Fig. 7: arabidopsis abca9 mutant and revertant phenotype；WT: wild-type Arabidopsis plants；OE-2: excess table Up to strain 2；2-C1:abca9-1 revertant strain 2；2-C2:abca9-2 revertant strain 2；2-C3:abca9-3 Revertant strain 2.

Fig. 8: the fat content of transgenosis, mutant and revertant plant seed；A in figure overexpresses plant seed Fat content；B in figure, mutant and revertant plant seed fat content.

Fig. 9: the content of fatty acid of transgenosis, mutant plants seed；A in figure, transgenic plant seed fatty acid content； B in figure, abca9 mutant plants seed fatty acid content.

Figure 10: the mass of 1000 kernel of overexpression, mutant and revertant plant seed；A in figure, transgenic seed thousand Weight；B in figure, mutant and revertant thousand grain weigth.

Specific embodiment

The clone of GmABCA9 gene

Using the black 44 kind RNA of agriculture of soybean as template, after synthesizing cDNA, pass through PCR amplification, amplimer pair are as follows:

5'-TGCTCTAGAATGGCAACCACTCTCACC-3'

5'-TGCTCTAGACCACTGAAAAGCTACTGAACT-3',

The gene order of 2904bp is obtained, as shown in SEQ ID No.1, is named as soybean GmABCA9 gene；Coding altogether 967 amino acid, as shown in SEQ ID No.2.

Expression analysis of the GmABCA9 in soyabean tissue

Verifying of the spatial and temporal expression of gene to a certain extent for purpose gene function provides important evidence.In order to analyze Expression of the GmABCA9 in soybean different tissues, we are extracted 44 soybean leaves of black agriculture of sowing 30d, root, stem, open The RNA for spending rear 30d seed and fruit pod detects expression of the GmABCA9 in soybean different tissues by real-time quantitative PCR (such as Fig. 1).

It is found after analyzing real-time quantitative PCR result, GmABCA9 expression quantity in the soyabean tissue of sowing 30d is equal It is lower than in the seed of Post flowering 30d, fruit pod.And in the seed and fruit pod for blooming 30d, the gene in seed expression quantity compared with It is high.The soybean of sowing 30d is in vegetative growth stage, and expression of the GmABCA9 in root, stem, leaf may be plant nutrition Growth provides nutriment.It is the generative growth phase of soybean plant strain from blooming to seed maturation, compared with fruit pod, Post flowering The expression quantity of the gene is higher in the seed of 30d, shows that GmABCA9 may be in the maturation of the soya seeds of Post flowering 30d In function, regulate and control the growth course of seed.

The subcellular localization of GmABCA9

In order to analyze the subcellular localization situation of GmABCA9 albumen, the GmABCA9 gene that we will clone in soybean, After removing terminator codon, is merged with the GFP of pCambia1300 label, construct 35S:GmABCA9-GFP carrier.Use limit Property restriction endonuclease BamHI processed carries out digestion verification, occurs the target fragment (such as Fig. 2) of 1523bp and 840bp as the result is shown, shows Vector construction success.

In order to analyze GmABCA9 subcellular localization situation, the 35S:GmABCA9-GFP carrier built is transferred to agriculture by us In bacillus GV3101, tender tobacco leaf is infected using Agrobacterium, the glimmering of GmABCA9 is observed under laser confocal microscope Optical signal (such as Fig. 3).In the micrograph of tobacco leaf, after infecting tobacco leaf using zero load, it can be observed in A in Fig. 3 To very strong green florescent signal, due to constructive expression of the 35S strong promoter in plant, it can be observed that many places fluorescence is believed Number, it is mainly distributed on nucleus, endoplasmic reticulum and cell membrane etc..When by the expression of 35S strong promoter starting GmABCA9 gene, (B in Fig. 3) has found that green florescent signal is looped around around nucleus in the tobacco leaf that 35S:GmABCA9-GFP infects, And it can be observed that apparent Filamentous signal, the red fluorescent with endoplasmic reticulum marker in C in Fig. 3 in cytoplasm Unanimously.By the gene green florescent signal it is Chong Die with endoplasmic reticulum marker danger signal after, in Fig. 3 in D it can be observed that Yellow signal shows that GmABCA9 is located in endoplasmic reticulum.

In order to exclude the interference of plant cell wall impurity and other materials to target gene fluorescence signal, we be extracted by The tobacco leaf protoplast infected, is observed under the microscope.It is similar with the signal in tobacco leaf, with comparative diagram 3 Middle E is compared, and the overlapping of F and G shows that GmABCA9 is located in endoplasmic reticulum in Fig. 3.

Expression of the GmABCA9 in transgenic arabidopsis

In order to verify the function of GmABCA9, we infect wildtype Arabidopsis thaliana by flower-dipping method, target gene are transferred to quasi- In southern mustard.T1 transgenic plant seed obtained is screened on the culture medium containing Hyg and the 1/2MS of Cef, is sieved altogether Choosing obtains 25 plants of positive seedlings.Transplanting extracts leaves genomic DNA and carries out PCR verifying after it grows up to healthy and strong seedling.Purpose band For 672bp, finally screen to 23 plants of positive plants (part amplification such as Fig. 4).

Expression of the GmABCA9 in arabidopsis

GmABCA9 higher expression in mature seed is had confirmed that in soybean, implies that the gene may participate in Regulate and control the growth course of seed.To verify expression pattern of the gene in Arabidopsis plant, we analyze GmABCA9 and are turning Expression in gene arabidopsis different tissues.Extract arabidopsis lotus throne leaf, stem leaf, root, the stem, Hua Hebo of sowing 30d The RNA of kind 40d silique, has detected expression of the GmABCA9 in transgenic arabidopsis different tissues by real-time quantitative PCR (such as Fig. 5).

It carries out analysis to real-time quantitative PCR result to find, GmABCA9 expression quantity highest in arabidopsis silique.Root, The gene expression amount is lower in stem, flower, lotus throne leaf and stem leaf, especially the most significant with stem leaf, and expression quantity is almost 0；And Root, stem, spending the expression quantity of the gene in three kinds of tissues, there is no apparent differences.In arabidopsis Seed development, silique Continuously needed nutrient matter is provided for seed.Therefore, great expression of the GmABCA9 in silique shows that the gene can The growth course of transgenic seed maturation can be taken part in.

The screening of soybean GmABCA9 gene reply arabidopsis abca9 mutant plants

The 35S:GmABCA9-GFP carrier built is transferred in Agrobacterium GV3101, infects arabidopsis using Agrobacterium Mutant plants.Target gene GmABCA9 is transferred in arabidopsis abca9 mutant, T is collected₁For revertant seed, It is screened on the culture medium of 1/2MS containing Hyg and Cef.Transplanting is extracted revertant and is planted after it grows up to healthy and strong seedling Strain leaves genomic DNA carries out PCR verifying, and the strain that purpose band is 672bp is that (part amplification is as schemed for positive plant 6).The result shows that in addition to strain 11, other strains are revertant plant in abca9-1-C plant.abca9-2-C Strain 1~14 with abca9-3-C plant is revertant plant.According to said method continue screening to T₃In generation, 3 kinds are returned Every kind of multiple mutant plants are total to screen 20 strains.

Arabidopsis abca9 mutant and the analysis of revertant seed phenotypes

Research report, the expression of arabidopsis AtABCA9 will affect the yield of seed grease.In order to verify the expression of the gene Whether the phenotype of arabidopsis seed will affect, we observe the seed phenotypes of arabidopsis abca9 mutant and revertant.

Under the microscope observe three kinds of abca9 mutant seeds phenotype (such as Fig. 7), find compared with WT, abca9-1 and The seed of abca9-2 mutant is shrivelled, and grain is small, and abca9-3 seed phenotypes and WT are almost consistent.When GmABCA9 is in wild type When with expressing in mutant plants, transgenic plant seed seed compared with WT is full and grain is big；And abca9-1 and abca9-2 Revertant seed is obviously fuller than mutant seeds, and kind grain becomes larger, especially with abca9-1 revertant seed phenotypes It is the most significant；But the phenotype and mutant of abca9-3 revertant seed have no apparent difference, it may be possible to because this is prominent The T-DNA insertion point of variant is located at the subregion that includes of the gene, does not completely inhibit the expression of the gene.As a result table Bright, the mutation of abca9 affects the turgor and kind grain size of seed.

Arabidopsis plant seed grease assay

By to three kinds of Arabidopsis Mutants and revertant seed phenotypes and without the growing state in sucrose culture medium After researching and analysing, it is subsequent that we have chosen phenotype more significant mutant abca9-1 and revertant abca9-1-C progress Functional study.In order to verify the function of GmABCA9, we have detected GmABCA9 transgenic plant, mutant and back mutation The content (such as Fig. 8) of body plant seed grease.Compared with WT plant, the seed oil of transgenic plant strain OE-1, OE-2, OE-4 Rouge content dramatically increases (A in such as Fig. 8), has increased separately 15%, 17%, 11%.Abca9-1 mutant seeds fat content with WT lines, which are compared, sharply to be declined.And when GmABCA9 overexpression in abca9-1 mutant, compared with mutant, return The fat content of multiple mutant strain 2-C1 and 3-C1 seed dramatically increases (B in such as Fig. 8), has increased separately 12% and 9%.Knot Fruit shows that the expression of GmABCA9 gene increases the content of Arabidopsis plant seed grease.

The measurement of Arabidopsis plant seed fatty acid content

In order to verify transgenic plant seed fat content increase whether by the increase of certain fatty acid caused by, I Have detected in seed the relative amounts (such as Fig. 9) of different fatty acid.Compared with WT lines, mutant plants seed C18: Although the relative amount of 2 and C18:3 increases, the content of C16:0 and C18:1 do not change (B in such as Fig. 9).And Sharply downward trend is but presented in abca9-1 mutant seeds total lipid content, shows that the mutation of abca9 may be raised or be lowered The level of other fatty acid reduces lipid total amount integrally.In transgenic plant OE-1, OE-2, OE-3 and OE-4 seed, The relative amount of C16:0, C18:1, C18:2 and C18:3 dramatically increase (A in such as Fig. 9).In OE-2 seed, C16:0, The relative amount of C18:1, C18:2 and C18:3 have increased separately 3.4mg/g, 11.4mg/g, 15.4mg/g and 10.2mg/g.Knot Fruit shows that GmABCA9 by the content of regulation Fatty Acids in Seeds, promotes the accumulation of seed grease.

The analysis of Arabidopsis plant thousand grain weigth

GmABCA9 gene expression influence Fatty Acids in Seeds and carbohydrate content, in order to verify the gene expression whether Influence whether the weight of seed, we have chosen the seed of 1000 transgenosis, mutant and back mutation body maturation, heavy to its Amount is detected.As a result, it has been found that (such as Figure 10).Compared with compareing 18mg, transgenosis OE-2 thousand grain weigth is 21mg, significant to increase Add 18%, and three kinds of mutant and the mass of 1000 kernel of revertant seed are consistent with wild type.The result shows that GmABCA9 mistake Amount expression increase seed weight, if by regulation Fatty Acids in Seeds and glucose content come influence seed weight need into One step research.

Regulate and control the synthesis of grease by above-mentioned experimental verification soybean transport protein GmABCA9.Fluorescence real-time quantitative PCR analysis It has been shown that, GmABCA9 is higher in soya seeds maturity period expression quantity, and seed maturity is the important period of seed oil and fat accumulation. GmABCA9 subcellular localization shows the assignment of genes gene mapping in endoplasmic reticulum and AtABCA9 positioning having the same.Arabidopsis abca9 is prominent Variant seed is shrivelled, and grain is small, and when growing in no sucrose culture medium, seedling yellow, develops abnormal.And GmABCA9 is returned For multiple mutant plants seed when no sucrose culture medium is grown, phenotype is normal.Arabidopsis abca9 mutant fat content is sharply Decline, the partial fatty acid constituent content for forming grease do not have significant change in abca9 mutant seeds, it may be possible to mutant In seed other fatty acid we do not analyze temporarily.GmABCA9 transgenic seed grease and fatty acid component content are significant Increase, shows that the expression of GmABCA9 gene increases the synthesis of grease by increasing the amount of Fatty Acids in Seeds.

Sequence table

<110>applicant's title Southwest University

<120>a kind of soybean lipid transport protein gene GmABCA9 and its application

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Leu Gly Arg Ser Gly Thr Val His Gly Val Val Ile Asp Pro Ser Gln

945 950 955 960

Val Ser Ser Val Ala Phe Gln

965

Claims (6)

1. soybean GmABCA9 gene, nucleotide sequence is as shown in SEQ ID No.1.

2. application of the soybean GmABCA9 gene as shown in SEQ ID No.1 in regulation vegetable seeds grease content.

3. application according to claim 2, which is characterized in that the plant is leguminous plant.

4. application according to claim 2, which is characterized in that the plant is soybean.

5. application according to claim 2, which is characterized in that the plant is arabidopsis.

6. the expression vector containing the soybean GmABCA9 gene as shown in SEQ ID No.1 is in plant seed lipid regulation Using.