CN103409528A - Method for discriminating output performance of royal jelly by using honeybee he*71 gene fluorescent quantitative PCR technology - Google Patents
Method for discriminating output performance of royal jelly by using honeybee he*71 gene fluorescent quantitative PCR technology Download PDFInfo
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Abstract
The invention relates to a method for discriminating output performance of royal jelly by using a honeybee he*71 gene fluorescent quantitative PCR technology. The method comprises steps of: acquisition of honeybee samples; extraction of head RNA of the honeybees; synthesis of the head cDNA of the honeybees; design of primers; qRT-PCR reaction system and condition; and discrimination of output performance of royal jelly. From the height of molecular biology, the invention provides a scientific and accurate fluorescent quantitative PCR method for discriminating output performance of royal jelly, aiming at the problem that production of royal jelly is critically influenced by conditions including bee colony potential, nectar source, environment and climate. In addition, the method can substantially shorten period for investigation of royal jelly production property, accelerate the speed of honeybee breeding, reduce cost of investigation of royal jelly production property and breeding cost, and mitigate working strength for field investigation of royal jelly output performance.
Description
Technical field
The present invention relates to a kind of method of utilizing fluorescent quantitative PCR technique to differentiate the royal jelly production performance, be specifically related to utilize honeybee storage protein (hex71) gene by fluorescence quantitative round pcr to differentiate the method for royal jelly production performance.
Background technology
China is bee-keeping big country, the major country of production of royal jelly especially, and the above royal jelly in the whole world 90% originates from China, and the Higher production royal jelly honeybee is the distinctive honeybee kind of China resource especially.All the time, the apiculture scientific worker of China is devoted to the correlative study of Higher production royal jelly heredity and breeding of honey bees, to the genetic marker of finding that the Higher production royal jelly proterties is relevant, is the service of honeybee royal jelly SOYBEAN IN HIGH-YIELD BREEDING.At honeybee Morphology level, cytology level, biochemistry level and molecular level, the relevant genetic marker of Higher production royal jelly is studied.Be summarized as follows:
The morphology genetic marker
Shao Ruiyi etc. (2003) find that worker bee is nose heave and have utmost point significant correlation with hypopharyngeal gland weight, and hypopharyngeal gland weight can be used as the important indicator of weighing the royal jelly production performance, and may there be certain dependency in head weight and the royal jelly production performance of therefore telling the slurry worker bee.Zheng Aijuan etc. (2010) find, slurry honeybee worker pupae nose heave each age in days all the utmost point to be significantly higher than original seed apis mellifera worker pupae nose heave, worker bee is nose heave likely becomes the externally mark on Morphology level of Higher production royal jelly honeybee kind.The honeybee hypopharyngeal gland is the main body of gland that synthesizes and secrete royal jelly that is positioned at Worker head, and its activity often is used to weigh the bleeding ability of honeybee.People study the anatomical morphology of hypopharyngeal gland and find, hypopharyngeal gland folliculus number, hypopharyngeal gland size and the weight of worker bee and the speed of the external synthetic protein of hypopharyngeal gland all can be used as the measurement index of hypopharyngeal gland activity, the dependency maximum of the hypopharyngeal gland length of worker bee and royal jelly output wherein, may be as the comparatively ideal morphology genetic marker of Apis mellifera royal jelly production performance.
The cytology genetic marker
By the karyotype to " Zhejiang Nongda No.1 " Apis mellifera drone and original seed Apis mellifera drone, analyze relatively and find, the 7th, 10,12 chromosomal arm ratios of two different honeybee kind drones exist utmost point significant difference.When this species diversity may be " Zhejiang Nongda No.1 " Apis mellifera drone chromosome duplication, control the multipair allelotrope fragment repeat replication that produces the slurry proterties, the cause that has caused the minor gene number to increase.In G-banding Analysis, also find the 3rd karyomit(e) " Zhejiang Nongda No.1 " Apis mellifera than the original seed Apis mellifera many a band.
Biochemical genetic marker
Isozyme is the multienzyme form produced by hereditary difference, can directly reflect the otherness of genetic expression, can be used as a kind of biochemical genetic marker and is widely used in the research that population genetic, molecular breeding and kind are identified.Malate dehydrogenase (malic acid dehydrogenase) (MDH) in honeybee research is the allozyme by three allelotrope codings, its heredity stabilizer pole.Its zymogram can be divided into San Ge district band: MDH I, MDH II, MDH III, but only have the MDH II to be polymorphism in type not at the same level and different developmental phases.By analyzing genotype frequency, gene frequency and the heterozygosis homozygosity of MDH II, aspect the biochemical genetic marker research of Higher production royal jelly honeybee kind, obtaining certain progress.Guan Yinghui etc. (1994) study discovery, the MDH II heterozygosity of Higher production royal jelly honeybee (" Zhejiang Nongda No.1 " Apis mellifera) is than the height of other two kinds of Apis melliferas (Hubei Apis mellifera and original seed apis mellifera), and new discovery unexistent aa, ab genotype in other two kinds of royal jelly low yield Apis melliferas, can be used as the biochemical genetic marker of Higher production royal jelly honeybee kind.In addition, all there are utmost point significant difference in the genotype frequency of the MDH II of three subspecies honeybees of apis mellifera, gene frequency and heterozygosis homozygosity, this result shows, can from the Biochemical Genetic angle, identify different honeybee kinds by gene frequency and the heterozygosis homozygosity of measuring the MDH II.Bao Xiuliang (1997) studies report, and Higher production royal jelly honeybee (" Zhejiang Nongda No.1 " Apis mellifera) and the MDH II polymorphism of other four kinds of Apis mellifera strains are significant difference.To sum up learn, the malate dehydrogenase (malic acid dehydrogenase) II of honeybee (MDH II) likely becomes the genetic marker of the biochemistry level that the Higher production royal jelly proterties is relevant.
Molecular genetic marker
In the certain progress obtained aspect microsatellite locus research, gene studies, still rare in the research of gene level at present, also need to use more ripe, more scientific technology to its further research.
Since in the genome honeybee, finding micro-satellite in 1993, the honeybee geneticist utilizes micro-satellite to carry out much research, these researchs mainly concentrate on attribute classification, origin, population genetic structural analysis and the genetic diversity Journal of Sex Research etc. on honeybee germ plasm resource, for the genetic breeding work of honeybee is laid a good foundation.There is the scholar to study discovery by adopting 10 microsatellite locus to produce the different honeybee (" Zhejiang Nongda No.1 " Apis mellifera, Italian Bee, original seed Apis mellifera) of slurry ability to 3 kinds, 10 microsatellite locus coamplification in 3 honeybee kinds goes out 96 allelotrope, comprise 48 difference allelotrope, show that namely these 10 microsatellite locus are the height polymorphism in 3 honeybee kinds, and have certain genetic distance between these 3 honeybee kinds.The analytical results of gene frequency shows, wherein 7 of 6 sites allelic frequencies are along with honeybee produces the grow of slurry ability and sequentially increases, and these 7 gene frequencies of " Zhejiang Nongda No.1 " Apis mellifera all are significantly higher than other two kinds of honeybees, simultaneously, 4 gene frequencies in other 4 sites present opposite trend, and namely the gene frequency of " Zhejiang Nongda No.1 " Apis mellifera is significantly lower than other two kinds of honeybees." Zhejiang Nongda No.1 " Apis mellifera, Italian Bee, original seed Apis mellifera are all the same subspecies that belong to apis mellifera, their product slurry performance difference mainly comes from the genetic diversity formed after artificial selection, by the Microsatellite DNA Analysis technology, three honeybee kinds studies have shown that to honeybee produces the 62%-89% that starches proterties and determined by inherited genetic factors, and the gene frequency of above-mentioned 10 microsatellite locus has presented the very diversity of rule in three are produced the different honeybee kind of slurry performance.Infer accordingly, may there be linkage relationship closely in the allelic frequency of these 10 microsatellite locus with the royal jelly output proterties.
1999, there is the people, from gene level, the Higher production royal jelly proterties has been carried out to desk study.W316bp is with random primer W(5 '-CGGCCCCGGT-3 '), by the specific DNA fragment that RAPD-PCR increases and obtains, numerous research shows that W316bp only appears in the polymorphism collection of illustrative plates of Higher production royal jelly Apis mellifera.Wang Weipings etc. (2002) utilize 12 kinds of random primers to carry out RAPD-PCR and Southern hybridization analysis to producing four race of bees that the slurry ability is different (" Zhejiang Nongda No.1 " Apis mellifera, Jiang Feng, Xiaoshan, Pinghu slurry honeybee, card honeybee), have obtained the relevant specific mark P2W316bp of Higher production royal jelly proterties.Zhang Yajuan etc. (2001) are prepared into probe by W316bp, with the RAPD-PCR amplified production of Higher production royal jelly honeybee and card honeybee, hybridize respectively, and result shows again, and W316bp may be the distinctive gene fragment of Higher production royal jelly Apis mellifera.
But also there is the scholar this to be proposed to query.They think, the honeybee kind of above experiment and sample size are very little, and sample is selected very science, can in the low yield honeybee, there is no because of in three Higher production royal jelly honeybees, having just simply to infer the mark that W316bp can be used as the Higher production royal jelly proterties, it is also likely relevant from the other biological characteristic of four different honeybee kinds.
Therefore, be necessary by more powerful maturation, the molecular studies technology of science is studied the Higher production royal jelly proterties from gene level more, to inquire into the method for differentiating honeybee royal jelly traits of yield from the molecular biology level.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the royal jelly production performance.Described honeybee hex71 gene, refer to honeybee storage protein (hex71) gene.
Order of the present invention is achieved through the following technical solutions.
A kind of method of utilizing honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the royal jelly production performance of the present invention is characterized in that discrimination method is as follows:
(1) honeybee sampling: from being investigated bee colony, every group gathers respectively 30 nurture honeybees, and described nurture honeybee refers to the worker bee of nurture larva in bee colony, the i.e. worker bee of 6-12 age in days;
(2) honeybee head RNA extraction and cDNA are synthetic: extract the RNA of gather nurture honeybee head, reverse transcription obtains cDNA; Namely adopt the TRizol method to extract RNA, adopt the reverse transcription test kit to complete the synthetic of cDNA;
(3) design of primers: the primer sequence that design is synthesized is as follows:
Primer-F：5’
- agtatggctggttggcttattg-3’
Primer-R：5’
- cgatgttggcttctatgttcc-3’；
(4) fluorescence quantitative PCR detection: the honeybee head cDNA of take after dilution is template, take Primer-F and Primer-R and is primer, carries out quantitative fluorescent PCR; The GAPDH that selects the honeybee head is reference gene, adopts 2
-△ △ ct
Method is calculated the expression amount of honeybee hex71 gene; In each qRT-PCR reaction of each bee colony sample, all repeat repeatedly, getting repeated mean value is the final expression amount of honeybee hex71 gene, and final expression amount is higher, shows that the royal jelly production performance of The bee colony is better; Described 2
-△ △ ctFor the relative expression quantity of honeybee hex71 gene, △ △ Ct=(Ct wherein
Target-Ct
GAPDH)
Treatment-(Ct
Target-Ct
GAPDH)
Control.
A kind of method of utilizing honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the royal jelly production performance of the present invention, the reaction system of its quantitative fluorescent PCR is: cumulative volume is 20ul, include SYBR Green I 10ul, Rox 0.4ul, Primer-F 0.4ul, Primer-R 0.4ul, cDNA 2ul, DEPC water is supplied 20ul.
A kind of method of utilizing honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the royal jelly production performance of the present invention, the reaction conditions of its quantitative fluorescent PCR is as follows: 95 ℃ of denaturations, 30s, PCR reacts 40 cycles, 95 ℃, 5s, 61 ℃, 31s, 55 ℃ of solubility curves, 20s.
Advantage of the present invention and beneficial effect: the present invention carries out deep research to screening honeybee hex71 gene, by the directive breeding means, in same honeybee kind, has cultivated respectively Higher production royal jelly and royal jelly low yield honeybee.According to 9792 honeybee genes that annotated from NCBI retrieval and the present invention's new 1722 genes that detect bee larva, and 175 genes that may be closely related with the royal jelly secretion, design and prepared the gene chip (Agilent) that contains 11689 honeybee genes, respectively with Higher production royal jelly honeybee and royal jelly low yield honeybee in telling the nurture honeybee of slurry head cDNA hybridize, preliminary screening goes out the gene of 369 differential expressions.In addition, gather the high and low product of royal jelly honeybee sample, by the qRT-PCR technology, difference expression gene is verified, filter out honeybee hex71 gene, as the relevant molecule marker of royal jelly production proterties.And then the design primer, the honeybee head cDNA of take is template, take Primer-F and Primer-R to be primer, carries out quantitative fluorescent PCR; The GAPDH that selects the honeybee head is reference gene, adopts 2
-△ △ ct
Method is calculated the expression level of honeybee hex71 gene; Its result is that the final expression amount of royal jelly production performance and honeybee hex71 gene is closely related, and the final expression amount of honeybee hex71 gene is high, and the royal jelly production performance is good.
Compared with prior art, remarkable advantage is in the present invention:
1, because royal jelly production is subjected to the condition influence such as bee colony group gesture, nectar source, environment, weather huge, the honeybee hex71 gene that the present invention adopts is as the relevant molecule marker of royal jelly high-yield character, can detect more scientific, exactly the quality of the royal jelly production traits of bee colony, for honeybee royal jelly SOYBEAN IN HIGH-YIELD BREEDING provides molecule assist-breeding means.
2, can detect easily in laboratory the royal jelly production traits of honeybee hex71 gene expression abundance with the judgement bee colony, alleviate the working strength that the field royal jelly production traits is investigated.
3, bee colony is from breeding to the time of royal jelly need of production more than two months, royal jelly production traits period of supervision is more than three months, and the detection of honeybee hex71 gene expression abundance only needs the time, so, adopt the present invention can greatly shorten royal jelly production traits period of supervision, accelerate breeding speed.
4, the inventive method is compared with traditional method, can greatly reduce cost and breeding cost that the royal jelly production traits is investigated.
Embodiment
The present invention is further elaborated below in conjunction with embodiment.
Embodiment 1
A kind of method of utilizing honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the royal jelly production performance comprises the following steps:
, the honeybee sample collection
1. produce and starch season, continuous 5 30 groups of honeybee tissues production royal jelly to Thousand-Island Lake, Zhejiang experiment bee farm, weighing is also recorded every group of each royal jelly weight and royal cells and is accepted quantity.
2. filter out respectively 5 Higher production royal jelly bee colonies and 5 royal jelly low yield bee colonies and other 4 royal jelly outputs between high yield and low yield and the royal jelly output bee colony of trend in gradient, totally 14 bee colonies are as being investigated bee colony.The royal jelly output scope of described Higher production royal jelly bee colony and royal jelly low yield bee colony is that three days a collection of group's product royal jelly of high yield group are not less than 100 grams, and three days a collection of groups of low yield group produce royal jelly not higher than 80 grams;
3. again organize selected bee colony to carry out royal jelly production, produce the second day of slurry, with tweezers 30 the nurture honeybees that telling slurry of collection of often mining massively respectively, put into immediately liquid nitrogen in 14 bee colonies, the tubulature in-80 ℃ of Refrigerator stores of then classifying.
2, RNA extracts
1. from Ultralow Temperature Freezer, taking out the honeybee sample, with the scissors after sterilizing, cut the head (in operation on ice) of honeybee, and with the PBS damping fluid of DEPC water preparation, wash away the dust of honeybee head.Described PBS is phosphoric acid buffer.
2. the honeybee head is put into to mortar, adds liquid nitrogen and be ground to powder, then by powder transfer to the 50ml centrifuge tube, in centrifuge tube, add TRizol, 30 honeybees add 3ml.With Syrup-homogenizing instrument or vibrator vibrate, homogenized.
3. the homogenate sample is placed to 10min in room temperature, the nucleic acid protein mixture is separated fully, and 4 ℃ of centrifugal 10min of 12000g, supernatant got.
4. getting supernatant is transferred to 3 1.5ml centrifuge tubes, and adds chloroform, vibrated 15 seconds, room temperature is placed 5min, and the add-on of described chloroform is every use 3ml TRizol, adds the 0.6ml chloroform.
5. 4 ℃ of centrifugal 15min of 12000g, get supernatant 500ul and be transferred in new 2ml centrifuge tube.
6. in supernatant liquor, add isopyknic Virahol (RNA in the precipitation water), mix rear room temperature and place 10min.
7. 4 ℃ of centrifugal 10min of 12000g.Gelatinous precipitate appears in pipe side and the pipe end, abandons supernatant (if the precipitation poor quality need, on the basis of this precipitation, add TRizol and carry out extracting again).
8. by volumetric concentration, be 75% ethanol (ice cool in advance, the preparation of DEPC water) washing RNA precipitation.Every use 3ml TRizol adds 3ml 75% ethanol at least, vibrate several under, be no more than the centrifugal 5min of 7500g, abandon supernatant.
9. drying at room temperature 10min, add 40ul DEPC water, inhales and beat several times with the rifle head, places 10min for 55 ℃, and RNA is dissolved.
10. Total RNA concentration determination: the concentration and the A260/A280 value that detect the RNA that extracts with NanoDrop 2000.
3, cDNA is synthetic
1. by 40 times (3ulRNA+117ulDEPC water) of above-mentioned satisfactory RNA dilution.
2. RNA thermally denature 5min under 65 ℃ of conditions, be put in immediately cooled on ice.
3. with the reverse transcription test kit, complete the synthetic of cDNA, configure following reaction solution (50ul system):
Total RNA, 0.6ul (usage quantity of annotating Total RNA is 500-1000ng); 5*RTbuffer, 10ul; Primer mix, 2.5 ul; RT Enzyme Mix, 2.5 ul; RNase free dH
2It is 50ul that O adds to total reaction volume.
4. reaction conditions: 37 ℃ of 15min(reverse transcription reactions)
98 ℃ of 5min (enzyme deactivation reaction)
5. take out and be placed in standbyly on ice, or be placed under-20 ℃ of conditions and preserve.
6. cDNA concentration determination: concentration and the A260/A280 value of measuring cDNA with NanoDrop 2000.
, design of primers
We have carried out the qRT-PC screening verification to honeybee storage protein (hex71) gene, and application primer5.0 and this gene primer of Primer3 Plus software design complete primer by Shanghai living work biotechnology company limited synthetic.Design altogether and synthesized 2 pairs of primers, comprising 1 checking gene and 1 reference gene GAPDH(control).Design and select the first principle of primer to be, at first guaranteeing the specificity of primer, can accurately amplify the purpose product; Next is that the size of amplified production is 80～250bp, because the amplified fragments in this scope relatively is applicable to the qRT-PCR method, amplified fragments is oversize can be reduced amplification efficiency and easily cause nonspecific reaction, the too short non-specific amplification that also can affect product of amplified fragments, affect quantitative accuracy; Finally, the GC content of the primer is in the 40-60% scope, and annealing temperature (Tm value) should be consistent, and is conducive to the setting of W-response condition.The primer sequence result is as shown in table 1.
The primer sequence of table 1 goal gene
5, qRT-PCR reaction system and condition
By 6 times of volume dilution of honeybee head cDNA of above-mentioned steps 3 gained (5ul stoste+25ul DEPC water), standby.And prepare following reaction system (20ul):
SYBR Green Ⅰ： 10ul
Rox： 0.4ul
Primer(F)： 0.4ul
Primer(R)： 0.4ul
cDNA： 2ul
DEPC water: 6.8ul
Reaction conditions:
95 ℃ of denaturations, 30s
PCR reacts (40 cycles)
95℃， 5s
61℃, 31s
55 ℃ of solubility curves, 20s
6, differentiate the royal jelly production performance
Selecting at the highly stable GAPDH of honeybee head expression is reference gene, adopts 2
-△ △ ctMethod is calculated the expression level of goal gene.Goal gene relative expression quantity=2 wherein
-△ △ Ct, △ △ Ct=(Ct
Target-Ct
GAPDH)
Treatment-(Ct
Target-Ct
GAPDH)
Control, selecting minimum that group honeybee of royal jelly output is control group (control), that other 13 groups of honeybees represent is experimental group (treatment).
Goal gene is equal triplicate in each qRT-PCR reaction of each bee colony sample, gets 2 of third-order reaction
-△ △ CtMean value is the final expression amount of this gene in this bee colony sample, and the final expression amount of honeybee hex71 gene is high, and the royal jelly production performance is good; Finally to the gene differential expression situation between the high and low product group of royal jelly honeybee sample (every group of 5 bee colonies), select the non-matching sample average t method of inspection in SPSS13.0 software to carry out statistical study, 0.01<P<0.05 o'clock, significant difference; P<0.01 o'clock, difference are extremely remarkable.
Embodiment 2, Higher production royal jelly honeybee hex71 gene expression abundance are significantly higher than royal jelly low yield honeybee
By continuous 5 royal jelly outputs to 30 groups of honeybees in bee farm statistics, 5 Higher production royal jelly bee colonies and 5 royal jelly low yield bee colonies have finally been screened.Result shows, the royal jelly mean yield that five groups of royal jelly low yield groups are five times is 64.32g, and the average royal jelly output that five groups of Higher production royal jelly groups are five times reaches 115.64g, and the results of analysis of variance that the royal jelly outputs of Higher production royal jelly, 10 groups of honeybees of low yield group carries out is shown to F
Isosorbide-5-Nitrae 0=76.3935, P=0.0001, i.e. it is extremely remarkable that the royal jelly output of the high and low product group of royal jelly existence is difference.
By the qRT-PCR technology, honeybee hex71 gene is carried out to the gene expression abundance detection.The gene expression abundance of the hex71 gene in five bee colony honeybees of Higher production royal jelly group is 11.6625 ± 5.459, the gene expression abundance of the hex71 gene in five bee colony honeybees of royal jelly low yield group is 2.797 ± 1.6883, through giving birth to system T-test, analyze discovery, the differential expression of honeybee hex71 gene in the high and low product group of royal jelly honeybee be (P=0.0008) significantly.
SEQUENCE LISTING
Sequence table
<110 > University Of Agriculture and Forestry In Fujian
<120 > utilize honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the method for royal jelly production performance
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213 > artificial sequence
<400> 1
agtatggctg gttggcttat tg 22
<210> 2
<211> 21
<212> DNA
<213 > artificial sequence
<400> 2
cgatgttggc ttctatgttc c 21
SEQUENCE LISTING
Sequence table
<110 > University Of Agriculture and Forestry In Fujian
<120 > utilize honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the method for royal jelly production performance
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213 > artificial sequence
<400> 1
agtatggctg gttggcttat tg 22
<210> 2
<211> 21
<212> DNA
<213 > artificial sequence
<400> 2
cgatgttggc ttctatgttc c 21
Claims (6)
1. method of utilizing honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the royal jelly production performance is characterized in that discrimination method is as follows:
(1) honeybee sampling: from being investigated bee colony, every group gathers respectively 30 nurture honeybees; Described nurture honeybee refers to the worker bee of nurture larva in bee colony, the i.e. worker bee of 6-12 age in days;
(2) honeybee head RNA extraction and cDNA are synthetic: extract the RNA of gather nurture honeybee head, reverse transcription obtains cDNA;
(3) design of primers: the primer sequence that design is synthesized is as follows:
Primer-F：5’
- agtatggctggttggcttattg - 3’
Primer-R：5’
- cgatgttggcttctatgttcc - 3’；
(4) fluorescence quantitative PCR detection: the honeybee head cDNA of take after dilution is template, take Primer-F and Primer-R and is primer, carries out quantitative fluorescent PCR; The GAPDH that selects the honeybee head is reference gene, adopts 2
-△ △ ct
Method is calculated the expression amount of honeybee hex71 gene; In each qRT-PCR reaction of each bee colony sample, all repeat repeatedly, getting repeated mean value is the final expression amount of honeybee hex71 gene, and final expression amount is higher, and the royal jelly production performance of The bee colony is better; Described 2
-△ △ ctFor the relative expression quantity of honeybee hex71 gene, △ △ Ct=(Ct wherein
Target-Ct
GAPDH)
Treatment-(Ct
Target-Ct
GAPDH)
Control.
2. a kind of method of utilizing honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the royal jelly production performance according to claim 1, the reaction system that it is characterized in that described quantitative fluorescent PCR is: cumulative volume is 20ul, include SYBR Green I 10ul, Rox 0.4ul, Primer-F 0.4ul, Primer-R 0.4ul, cDNA 2ul, DEPC water is supplied 20ul.
3. a kind of method of utilizing honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the royal jelly production performance according to claim 1, the reaction conditions that it is characterized in that described quantitative fluorescent PCR is as follows: 95 ℃ of denaturations, 30s, PCR reacts 40 cycles, 95 ℃, 5s, 61 ℃, 31s, 55 ℃ of solubility curves, 20s.
4. a kind of method of utilizing honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the royal jelly production performance according to claim 1, it is characterized in that described honeybee head RNA extracts and cDNA is synthetic, adopt the TRizol method to extract RNA, adopt the reverse transcription test kit to complete the synthetic of cDNA.
5. a kind of method of utilizing honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the royal jelly production performance according to claim 1, is characterized in that the honeybee head cDNA after described dilution, namely with DEPC water by 6 times of volumes of honeybee head cDNA dilution.
6. a kind of method of utilizing honeybee hex71 gene by fluorescence quantitative round pcr to differentiate the royal jelly production performance according to claim 1, is characterized in that described each qRT-PCR at each bee colony sample all repeats repeatedly in reacting, and its multiplicity is 3 times.
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| CN103937902A (en) * | 2014-05-07 | 2014-07-23 | 浙江大学 | Method for detecting output of bee milk by bee verm gene |
| CN103937903A (en) * | 2014-05-07 | 2014-07-23 | 浙江大学 | Method for detecting yield of royal jelly by using bee LOC726515 gene |
| CN103952489A (en) * | 2014-05-07 | 2014-07-30 | 浙江大学 | Method of detecting yield of royal jelly by LOC409360 gene of bee |
| CN104988240A (en) * | 2015-08-03 | 2015-10-21 | 福建农林大学 | Method for identifying swarm royal jelly high yield character with SNP mark rs16287910 |
| CN104988240B (en) * | 2015-08-03 | 2017-12-08 | 福建农林大学 | Differentiate the method for bee colony Higher production royal jelly character using SNP marker rs16287910 |
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