CN106282364B - Two high oleic acid rape sldh genes and its screening technique and application - Google Patents
Two high oleic acid rape sldh genes and its screening technique and application Download PDFInfo
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- CN106282364B CN106282364B CN201610801322.5A CN201610801322A CN106282364B CN 106282364 B CN106282364 B CN 106282364B CN 201610801322 A CN201610801322 A CN 201610801322A CN 106282364 B CN106282364 B CN 106282364B
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Abstract
The invention belongs to field of biotechnology, are related to the breeding of high oleic acid rape, and in particular to two high oleic acid rape sldh genes and its screening technique and application.Two kinds of high oleic acid rape sldh genes are atpB gene and cretinephosphokinase gene.The screening technique of the gene, steps are as follows: according to transcriptome analysis, screening the candidate gene of high oleic acid rapeseed breeding material early screening；The different different growing rape material of oleic acid content is acquired, the differential expression amount of candidate gene is measured；The fatty acid composition of rape mature seed is measured by gas chromatography；Differential expression amount is compared with fatty acid composition, screens high oleic acid rapeseed gene.A large amount of unknown materials are screened, large quantities of breeding materials for there are high oleic acid potentiality are obtained, carry out later period research, accelerate breeding process, promote the breeding of high oleic acid new rape variety.
Description
Technical field
The invention belongs to field of biotechnology, are related to the breeding of high oleic acid rape, and in particular to two high oleic acid rape mirror
Other gene and its screening technique and application.
Background technique
High oleic acid rape oil has good alimentary health-care function, and nutritive value can compare favourably with olive oil, tea oil, thus its
Large-scale promotion plays a very important role for the optimization and upgrading of Rape industry and the edible oil level of improvement our people.Together
When, the benefit at rapeseed cultivation family can also be increased, effectively increase Rape-seed production, promote the extensive development of China's Rape industry, protected
Hinder China's edible oil safety.
Although selecting some kinds both at home and abroad at present, but still it is difficult to meet growing wilderness demand, traces it to its cause
Mainly high oleic acid new rape variety breeding multi-pass crosses hybridization or directive breeding technology obtains material, in field planting, after mature
It is analyzed using seed of the gas chromatograph to harvest, high-oleic acid material is determined whether it is according to result.This method is time-consuming
(often it is only a season) long, and field work management and post analysis detection work are more heavy, breeding of new variety difficulty is big；Together
When, it also needs to put into a large amount of human resources, increases breeding cost, be unfavorable for new varieties large-scale promotion；Therefore using a kind of
Simply, quickly screening technique come determine high oleic acid rape material become current high oleic acid rapeseed breeding research key.
In the research of early screening high oleic acid rapeseed breeding material, such as Wang Jingqiao is placed in by observation containing sodium hypochlorite
Identify high oleic acid mutant in early stage with leaf oxidation bleaches in the mixed solution of tween speed.The discoveries such as Gao Jianqin remove
Outside florescence, the oleic acid relative amount in each breeding time nutrition organs and reproductive organs is in extremely significant positive correlation.Noelle A etc. is logical
It crosses fast using FAD2 gene expression amount in real time fluorescence quantifying PCR method detection high oleic acid peanut and its wild type seeds and blade
Speed detection high oleic acid peanut genotype.These methods can reduce the cumbersome work in breeding, but without carry out systematic research and
Subsequent authentication, therefore, it is difficult to apply in production.
Summary of the invention
The present invention is to solve intricate operation in rapeseed breeding, and the long technical problem of breeding cycle provides two high oleic acids
Rape sldh gene and its screening technique and application.
In order to solve the above technical problems, the invention adopts the following technical scheme:
Two kinds of high oleic acid rape sldh genes, the high oleic acid rape sldh gene 1 are atpB gene, sequence such as SEQ
Described in ID NO:1；The high oleic acid rape sldh gene 2 is cretinephosphokinase gene, sequence such as SEQ ID NO:2 institute
It states.
The screening technique of two high oleic acid rape sldh genes, steps are as follows:
(1) according to transcriptome analysis, the candidate gene of high oleic acid rapeseed breeding material early screening is screened；
(2) the different different growing rape material of acquisition oleic acid content, measures the differential expression amount of candidate gene；
(3) pass through the fatty acid composition of rape mature seed in gas chromatography determination step (2)；
(4) the differential expression amount in step (2) is compared with the fatty acid composition in step (3), screens high oleic acid
Rapeseed gene.
Application of the screening technique of two high oleic acid rape sldh genes as screening high oleic acid rapeseed breeding material.
The beneficial effects of the present invention are:
1. carrying out Effective selection in rapeseed breeding material vegetative growth phase, reducing a large amount of cumbersome later period field management and dividing
Analysis detection work, reduces breeding cost.
2. a pair a large amount of unknown materials screen, large quantities of breeding materials for there are high oleic acid potentiality are obtained, later period research is carried out,
Accelerate breeding process, promotes the breeding of high oleic acid new rape variety.
Detailed description of the invention
Fig. 1 is the expression quantity of atpB gene different times in high oleic acid rape and low oleic acid rape.
Fig. 2 is the expression quantity of cretinephosphokinase gene different times in high oleic acid rape and low oleic acid rape.
Fig. 3 is expression quantity of the atpB gene in the rape of different oleic acid contents.
Fig. 4 is expression quantity of the cretinephosphokinase gene in the rape of different oleic acid contents.
Fig. 5 is expression quantity of the atpB gene in hybrid seed is emerged 7 days.
Fig. 6 is expression quantity of the atpB gene in the hybrid seed 5-6 leaf phase.
Fig. 7 is expression quantity of the atpB gene in hybrid seed Wintering Period.
Fig. 8 is expression quantity of the cretinephosphokinase gene in hybrid seed is emerged 7 days.
Fig. 9 is expression quantity of the cretinephosphokinase gene in the hybrid seed 5-6 leaf phase.
Figure 10 is expression quantity of the cretinephosphokinase gene in hybrid seed Wintering Period.
Specific embodiment
1. material prepares
High oleic acid rape material, Hunan oil 15 and different oleic acid content materials improve center Hunan branch center by national oil plant
It provides.
2. test method
2.1 quantitative PCR detection
Sample preparation: 7d takes 10 plants after emergence, and 5-6 leaf phase (at the beginning of 12 months) and Wintering Period (or so New Year's Day) respectively take 10 plants of oil
Dish tender leaf (1 piece/plant) is spare, and the florescence (mid-March) respectively takes the flower (3 piece/plant) of 10 plants of selfing baggings, (4 20-35d after pollination
The middle of the month) respectively take the immature seed (0.5g/ plants) of 10 plants of selfings and its silique skin (3/plant) mixing to be stored in -80 DEG C.
RNA is extracted: being extracted total serum IgE and is used TransZol Up Plus RNA Kit (Trans GeneBiotech), specifically
Method is referring to specification.It is detected through 2000 micro ultraviolet specrophotometer (Thermo) of Nanodrop and agarose gel electrophoresis
Meet the requirements, be stored in -80 DEG C it is spare.
Quantitative PCR analysis:
(1) it is analyzed according to transcript profile result (by Hua Da gene on behalf of progress), table in two gene different growing materials
Up to situation analysis；
(2) two genes expression in 56%, 65%, 75% and 81% rape 5-6 leaf phase blade is analyzed, as a result such as
Shown in Fig. 3；
(3) high-oleic acid material (oleic acid content 81.4%) and Hunan oil 15 (oleic acid content 62.5%) filial generation different bearing
The differential gene expression situation analysis of phase.
Reverse transcription reaction uses TransScript All-in-One First-Strand cDNA (Trans
GeneBiotech), specific method is referring to specification.Primer is designed using Primer 5.0, is closed by Shanghai Sheng Gong technology company
At.Real-time fluorescence quantitative PCR is carried out using two one-step circulation methods using CFX96TM Real-Time System (BIO-RAD, USA)
Reaction, reaction fluorescence signal carry out signal collection and analysis by CFX manager software program.Reaction condition
Are as follows: 95 DEG C of initial denaturation 30s subsequently enter 40 circulations, and 95 DEG C of denaturation 5s in each circulation, last 60 DEG C of annealing 30s, 72 DEG C are prolonged
Stretch 15s.UBC21 (ubiquitin-conjugating enzyme 21) is used as reference gene, upstream primer sequence such as SEQ ID
Shown in NO:7, downstream primer sequence is as shown in SEQ ID NO:8.Expression conditions are commented using compares cycle threshold method.
Data analysis uses ANOVA and Post Hoc test, and p value < 0.05 indicates that there are significant differences.
2.2 rape seed attributional analysis
Laboratory apparatus: SP-6890 type gas chromatograph, fid detector and N3000 chromatographic work station capillary chromatographic column
DB-23 (Anjelen Sci. & Tech. Inc)；AL204 electronic balance (Mei Tele-support benefit Internaional, Inc).
Chromatographic condition: 270 DEG C of injector temperature；190 DEG C of column temperature；230 DEG C are risen to 5 DEG C/min, keeps 1min；Detector
260 DEG C of temperature；Nitrogen flow 250mLmin-1；Hydrogen flowing quantity 30mLmin-1；Air mass flow 300mLmin-1；Split ratio
40:1；Column flow 1.0mLmin-1；Sample volume: 1.0 μ L.
Sample preparation and measurement: taking 0.2g baked seed, crushed with high speed disintegrator, is placed in 10mL test tube with ground stopper, then
The ether-petroleum ether that 2mL volume ratio is 1:1 is added to be added in 10mL test tube, the KOH- methanol that 1mL 0.5M is added is catalyzed
Test tube is placed in 40 DEG C of water bath with thermostatic control by agent, and after reaction for 24 hours, static layering is sampled from upper layer.
3. result and analysis
Expression is analyzed in 3.1 two gene different growing materials
The different rape different growing material of oleic acid content is acquired, and measures candidate differential gene expression amount；AtpB base
The expression quantity of cause is as shown in Figure 1 with primer pair SEQ ID NO:3 and SEQ ID NO:4 progress fluorescent quantitative PCR result；Creatine phosphorus
The expression quantity of kinase gene carry out fluorescent quantitative PCR result as schemed with primer pair SEQ ID NO:5 and SEQ ID NO:6
Shown in 2.According to result it is found that this 2 genes have evident regularity in rape vegetative growth phase (before the florescence) expression, can be used for into one
Step research.
3.2 two genes expression in 56%, 65%, 75% and 81% rape 5-6 leaf phase blade is analyzed
It acquires oleic acid content and is 56%, 65%, 75% and 81% rape 5-6 leaf phase blade, and measure candidate difference base
Because of expression quantity；The expression quantity of atpB gene carries out fluorescent quantitative PCR result with primer pair SEQ ID NO:3 and SEQ ID NO:4
As shown in Figure 3；The expression quantity of cretinephosphokinase gene is carried out glimmering with primer pair SEQ ID NO:5 and SEQ ID NO:6
Fluorescent Quantitative PCR result is as shown in Figure 4.According to result it is found that this 2 genes are in different oleic acid content rape 5-6 leaf phase blades
Expression compares obvious relation between persistence between oleic acid content, can predict rape oleic acid content according to expression conditions.
3.3 high-oleic acid materials (oleic acid content 81.4%) and Hunan oil 15 (oleic acid content 62.5%) filial generation gene expression
Situation analysis
7d, 5-6 leaf phase and Wintering Period material after filial generation difference is emerged are acquired, and measures candidate differential gene expression
Amount；The expression quantity of atpB gene carries out fluorescent quantitative PCR result such as Fig. 5-7 with primer pair SEQ ID NO:3 and SEQ ID NO:4
It is shown；The expression quantity of cretinephosphokinase gene carries out carry out fluorescent quantitation with primer pair SEQ ID NO:5 and SEQ ID NO:6
PCR result is as seen in figs. 8-10.
3.4 high-oleic acid materials (oleic acid content 81.4%) and Hunan oil 15 (oleic acid content 62.5%) filial generation attributional analysis
Using the fatty acid composition of gas chromatography measurement filial generation mature seed, specific method is referring to 2.2, as a result such as
Table 1, from the above results, filial generation are mainly distributed on tri- regions 60-63,69-73 and 78-82, wherein the region 69-76
At most, account for about 50%, meet Mendel's regulavity of segregation.
21 filial generation seed oleic acid contents (Hunan 15 oleic acid contents 62.1% of oil, high oleic acid rape seed oleic acid contents
80.5%)
Table 1
Comparison gene expression results it is found that this 2 genes different filial generation vegetative growth phase expressions compare with
There is obvious relation between persistence between oleic acid content, can be used for early screening high oleic acid rapeseed breeding material.
3.5 result
It is final to determine that atpB gene and cretinephosphokinase gene can be used for high oleic acid rapeseed breeding material early screening, it sieves
The atpB gene selected, for gene order as shown in SEQ ID NO:1, the enzyme of the gene expression is located at thylakoid membrane, matter
On film or mitochondrial inner membrane, participates in oxidative phosphorylation and reacted with photophosphorylation, there is oxidative phosphorylation, light and system electronic to turn
The effects of shifting, catalyzes and synthesizes energy " currency " --- the ATP of organism under the promotion of cross-film proton dynamics.
Cretinephosphokinase gene, gene order is as shown in SEQ ID NO:2, in metabolism (carbon metablism, carbohydrate
Metabolism, energetic supersession), transhipment (phosphorus-containing groups, the transhipment of pairs of receptor phosphorylcholine group, phosphokinase) etc. plays in physiology courses and focuses on
Big effect.
Claims (1)
- Application of the 1.atpB gene in screening high oleic acid rapeseed breeding material, the atpB gene order such as SEQ ID NO.1 It is shown.
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