CN103409528A - Method for discriminating output performance of royal jelly by using honeybee he*71 gene fluorescent quantitative PCR technology - Google Patents

Abstract

The invention relates to a method for identifying the yield performance of royal jelly by utilizing the fluorescent quantitative PCR technology of the bee hex71 gene, including honeybee sample collection, honeybee head RNA extraction, honeybee head cDNA synthesis, primer design, qRT-PCR reaction system and conditions, and royal jelly Yield performance identification. From the perspective of molecular biology, the present invention aims at the problem that royal jelly production is greatly affected by conditions such as bee colony potential, nectar source, environment, and climate, and provides a fluorescent quantitative PCR that can more scientifically and accurately identify the pros and cons of royal jelly production performance of bee colonies The method can greatly shorten the inspection cycle of royal jelly production traits, speed up bee breeding, reduce the cost of royal jelly production trait inspection and breeding costs, and reduce the work intensity of field royal jelly yield performance inspection.

Description

A method for identifying the yield performance of royal jelly by using the fluorescent quantitative PCR technology of honeybee hex71 gene

technical field

The invention relates to a method for identifying the yield performance of royal jelly by using fluorescent quantitative PCR technology, in particular to a method for identifying the yield performance of royal jelly by using the fluorescent quantitative PCR technology of honeybee storage protein (hex71). the

Background technique

my country is a big beekeeping country, and it is also a major producer of royal jelly. More than 90% of the world's royal jelly is produced in China, and royal jelly-yielding bees are a unique bee resource in my country. For a long time, my country's bee industry scientific and technological workers have been committed to the related research on the genetic breeding of bees with high royal jelly yield, in order to find the genetic markers related to the high-yield traits of royal jelly, and serve the high-yield breeding of bee royal jelly. The genetic markers related to the high yield of royal jelly were studied at the level of morphology, cytology, biochemistry and molecular level. An overview is as follows:

Morphological genetic markers

Shao Ruiyi et al. (2003) found that there is a significant correlation between the head weight of worker bees and the weight of hypopharyngeal glands, and the weight of hypopharyngeal glands can be used as an important indicator to measure the production performance of royal jelly. Therefore, there may be a certain relationship between the head weight of worker bees and the production performance of royal jelly. Correlation. Zheng Aijuan et al. (2010) found that the head weight of worker bee pupae was significantly higher than that of the original species Apis mellifera at all ages, and worker bee head weight may be a marker of high royal jelly-producing bee species at the external morphological level . The honeybee hypopharyngeal gland is the main gland located in the head of the worker bee that synthesizes and secretes royal jelly, and its activity is often used to measure the bee's ability to secrete royal jelly. People have studied the anatomy and morphology of the hypopharyngeal glands and found that the number of hypopharyngeal gland sacs, the size and weight of the hypopharyngeal glands, and the rate of protein synthesis outside the hypopharyngeal glands of worker bees can all be used as indicators of hypopharyngeal gland activity. The length of the hypopharyngeal gland has the greatest correlation with the production of royal jelly, and it may be used as a morphological genetic marker for the ideal production performance of royal jelly in Italy.

cytology genetic markers

Through the analysis and comparison of the chromosome karyotypes of the "Zhenongda No. 1" Italian drone and the original Italian drone, it was found that there were extremely significant differences in the arm ratios of chromosomes 7, 10, and 12 of the drones of the two different bee species. This difference may be due to the repeated duplication of multiple pairs of allelic fragments that control the traits of pulp production during the chromosome replication of the "Zhenongda No. 1" Italian bee drone, resulting in an increase in the number of minor genes. Chromosome G-band analysis also found that the third chromosome "Zhenongda No. 1" Italian bee had one more band than the original species.

biochemical genetic marker

Isozymes are multi-enzyme forms produced by genetic differences, which can directly reflect the differences in gene expression, and can be widely used as a biochemical genetic marker in the research of population genetics, molecular breeding and species identification. Malate dehydrogenase (MDH) in honeybee studies is an allelic enzyme encoded by three alleles and is extremely heritably stable. Its zymogram can be divided into three zones: MDHⅠ, MDHⅡ, and MDHⅢ, but only MDHⅡ is polymorphic in different grades and different developmental stages. By analyzing the genotype frequency, gene frequency and heterozygosity of MDHIⅡ, some progress has been made in the study of biochemical genetic markers of royal jelly-producing bee species. Guan Yinghui et al. (1994) found that the MDHⅡ heterozygosity of the high-yield royal jelly bee (“Zhenongda No. 1”) was higher than that of the other two types of honeybees (Hubei The aa and ab genotypes that do not exist in the royal jelly-yielding bee species can be used as biochemical genetic markers for the royal jelly-yielding bee species. In addition, there are extremely significant differences in the genotype frequency, gene frequency and degree of heterozygosity of MDHⅡ among the three subspecies of Apis mellifera. Identification of different bee species. Bao Xiuliang (1997) reported that the MDHⅡ polymorphisms of high-yield royal jelly bees (“Zhenongda No. 1” Italian bee) and other four Italian bee strains were significantly different. In summary, malate dehydrogenase Ⅱ (MDH Ⅱ) in bees may be a biochemical genetic marker related to the high-yield traits of royal jelly.

molecular genetic marker

At present, some progress has been made in microsatellite site research and gene research, but research at the gene level is rare, and more mature and scientific techniques are needed for further research.

Since the discovery of microsatellites in the honeybee genome in 1993, honeybee geneticists have conducted many studies using microsatellites, which have mainly focused on quality classification, origin, population genetic structure analysis, and genetic diversity on honeybee germplasm resources. Research, etc., laid the foundation for the genetic breeding of bees. Some scholars have used 10 microsatellite loci to study 3 kinds of bees with different pulp production ability ("Zhe Nong Da No. 1" Italian bee, local Italian bee, and original species of Italian bee) A total of 96 alleles were amplified from the three bee species, including 48 differential alleles, which indicated that these 10 microsatellite loci were highly polymorphic in the three bee species, and the differences between the three bee species There is a certain genetic distance. The results of allele frequency analysis showed that the frequency of 7 alleles at 6 sites increased sequentially with the increase of bee pulp production ability, and the 7 allele frequencies of "Zhenongda No. 1" Italian bee Allele frequencies were significantly higher than those of the other two bees. At the same time, the 4 allele frequencies of the other 4 loci showed the opposite trend, that is, the allele frequencies of "Zhenongda No. 1" bees were significantly lower than those of the other two bees. Kind of bees. "Zhejiang Nongda No. 1" Italian bee, local Italian bee, and original species of Italian bee belong to the same subspecies of Apis mellifera, and the difference in their pulp production performance is mainly due to the genetic diversity formed after artificial selection. Microsatellite DNA analysis Technical research on three bee species has proved that 62%-89% of the bee's pulp production traits are determined by genetic factors, and the allele frequencies of the above 10 microsatellite loci are the same among the three bee species with different pulp production performance. There is a very regular diversity. Accordingly, it was speculated that the allele frequencies of these 10 microsatellite loci may be closely linked with the yield traits of royal jelly. the

In 1999, someone conducted a preliminary exploration of the high-yield traits of royal jelly from the gene level. W316bp is a specific DNA fragment obtained by RAPD-PCR amplification with random primer W (5'—CGGCCCCGGT—3’). Numerous studies have shown that W316bp only appears in the polymorphism map of royal jelly high-yielding bees. Wang Weiping (2002) used 12 kinds of random primers to conduct RAPD-PCR and Southern hybridization on four bee lines with different pulp production abilities ("Zhenongda No. 1" Italian bee, Pinghu bee, Xiaoshan bee, and card bee) Through the analysis, the specific marker P2W316bp related to the high-yield traits of royal jelly was obtained. Zhang Yajuan et al. (2001) prepared W316bp as a probe and hybridized it with RAPD-PCR amplification products of royal jelly high-yielding bees and card bees respectively. The results again showed that W316bp may be a unique gene fragment of royal jelly high-yielding bees. the

But some scholars have questioned this. They believe that the number of bee species and sample size in the above experiment is too small, and the sample selection is not very scientific. It cannot be simply inferred that W316bp can be used as a marker of high-yielding royal jelly just because three bees with high-yielding royal jelly have it but not among the low-yielding bees. There may be other biological properties related to the four different bee species. the

Therefore, it is necessary to study the high-yield traits of royal jelly from the gene level with the help of more powerful, mature and scientific molecular research techniques, so as to explore the method of identifying the yield performance of bee royal jelly from the molecular biology level. the

Contents of the invention

The purpose of the present invention is to provide a method for identifying the yield performance of royal jelly by using the honeybee hex71 gene fluorescent quantitative PCR technology. The honeybee hex71 gene refers to the honeybee storage protein (hex71) gene. the

The object of the present invention is achieved through the following technical solutions. the

A kind of method of utilizing honeybee hex71 gene fluorescent quantitative PCR technique of the present invention to discriminate royal jelly yield performance, it is characterized in that discriminating method is as follows:

(1) Honey bee sampling: 30 nurse bees were collected from each colony under investigation, and the nurse bees refer to worker bees that feed larvae in the bee colony, that is, worker bees that are 6-12 days old;

(2) RNA extraction and cDNA synthesis from the bee head: extract the RNA from the head of the breeding bee collected, and reverse transcribe to obtain cDNA; that is, use the TRizol method to extract RNA, and use the reverse transcription kit to complete the cDNA synthesis;

(3) Primer design: The designed and synthesized primer sequences are as follows:

Primer-F: 5' - agtatggctggttggcttattg-3'

Primer-R: 5' - cgatgttggcttctatgttcc-3';

(4) Fluorescence quantitative PCR detection: use the diluted bee head cDNA as a template, and use Primer-F and Primer-R as primers to perform fluorescence quantitative PCR; select GAPDH from the bee head as an internal reference gene, and use 2 ^{- △△ The ct} method is used to calculate the expression level of the bee hex71 gene; it is repeated several times in each qRT-PCR reaction of each bee colony sample, and the average value of multiple repetitions is taken as the final expression level of the honeybee hex71 gene, and the higher the final expression level , it indicates that the royal jelly production performance of the bee colony is better; the 2 ^{- △ △ ct} is the relative expression level of the bee hex71 gene, where △ △ Ct = (Ct _target -Ct _GAPDH ) _treatment - (Ct _target -Ct _GAPDH ) _control .

A kind of method of the present invention utilizes honeybee hex71 gene fluorescent quantitative PCR technology to identify the yield performance of royal jelly, the reaction system of its fluorescent quantitative PCR is: total volume is 20ul, contains SYBR GreenⅠ 10ul, Rox 0.4ul, Primer-F 0.4ul, Primer-R 0.4ul, cDNA 2ul, DEPC water to make up to 20ul. the

A method for identifying the yield performance of royal jelly by using the fluorescent quantitative PCR technology of honey bee hex71 gene of the present invention, the reaction conditions of the fluorescent quantitative PCR are as follows: pre-denaturation 95°C, 30s, PCR reaction 40 cycles, 95°C, 5s, 61°C, 31s , melting curve 55℃, 20s. the

Advantages and beneficial effects of the present invention: the present invention conducts in-depth research on the screening of the honey bee hex71 gene, and breeds bees with high royal jelly production and low royal jelly production respectively in the same bee species by means of directional selection. Based on the 9792 annotated bee genes retrieved from NCBI and the 1722 newly detected genes in bee larvae of the present invention, as well as 175 genes that may be closely related to royal jelly secretion, a gene containing 11689 bee genes was designed and prepared The chip (Agilent) was hybridized with the head cDNA of nurse bees that were producing jelly in high-yield royal jelly bees and low-yield royal jelly bees, and 369 differentially expressed genes were preliminarily screened out. In addition, samples of bees with high and low royal jelly yields were collected, differentially expressed genes were verified by qRT-PCR technology, and the bee hex71 gene was screened out as a molecular marker related to royal jelly yield traits. Then design primers, use bee head cDNA as template, Primer-F and Primer-R as primers, carry out fluorescent quantitative PCR; select GAPDH of bee head as internal reference gene, and use 2 ^{- △△ct} method to calculate honeybee hex71 gene The expression level; the result is that the yield performance of royal jelly is closely related to the final expression level of the bee hex71 gene, and the higher the final expression level of the bee hex71 gene, the better the yield performance of royal jelly.

Compared with the prior art, the present invention has significant advantages:

1. Because the production of royal jelly is greatly affected by conditions such as bee colony potential, nectar source, environment, and climate, the honeybee hex71 gene used in the present invention is used as a molecular marker related to the high-yield traits of royal jelly, which can more scientifically and accurately detect the royal jelly production traits of bee colonies The advantages and disadvantages of the honeybee royal jelly provide molecular assisted breeding methods for high-yield breeding of honeybee royal jelly.

2. The expression abundance of the bee hex71 gene can be conveniently detected in the laboratory to judge the royal jelly production traits of the bee colony, and reduce the work intensity of field investigation of royal jelly production traits. the

3. It takes more than two months for bee colonies to produce royal jelly, and the inspection period for the production of royal jelly is more than three months, while the detection of the abundance of bee hex71 gene expression only takes one day. Therefore, the use of the present invention can greatly shorten the production of royal jelly. Production traits inspection cycle to speed up breeding. the

4. Compared with the traditional method, the method of the present invention can greatly reduce the cost of inspecting the production traits of royal jelly and the cost of breeding. the

Detailed ways

    Below in conjunction with embodiment the present invention is further elaborated.

Example 1  A method utilizing honeybee hex71 gene fluorescent quantitative PCR technology to identify the yield performance of royal jelly, comprising the following steps:

, bee sample collection

① During the jelly production season, 30 colonies of bees in Zhejiang Qiandao Lake Experimental Bee Farm were organized to produce royal jelly for 5 consecutive times, and the weight of royal jelly and the amount of royal jelly received by each colony were weighed and recorded.

②5 colonies with high royal jelly production, 5 colonies with low royal jelly production, and 4 other bee colonies whose royal jelly production was between high and low yields with a gradient trend were screened out, and a total of 14 bee colonies were selected as the investigated bee colonies. The royal jelly production range of the royal jelly high-yield bee colony and the royal jelly low-yield bee colony is that the high-yield colony produces no less than 100 grams of royal jelly per three-day group, and the low-yield colony produces no more than 80 grams of royal jelly per three-day group;

③ Organize the selected bee colonies again to produce royal jelly. On the second day of production, use tweezers to collect 30 nurse bees that are spit out in each of the 14 bee colonies, put them into liquid nitrogen immediately, and then sort them into tubes and stored in -80°C refrigerator.

2. RNA extraction

① Take out the bee sample from the ultra-low temperature refrigerator, cut off the head of the bee with sterilized scissors (operate on ice), and wash off the dust on the head of the bee with PBS buffer solution prepared with DEPC water. The PBS is a phosphate buffer.

②Put the bee head into a mortar, add liquid nitrogen and grind it into powder, then transfer the powder to a 50ml centrifuge tube, add TRizol to the centrifuge tube, add 3ml to 30 bees. Shake and homogenize with a homogenizer or shaker. the

③Place the homogenized sample at room temperature for 10 minutes to completely separate the nucleic acid-protein complex, and centrifuge at 12,000 g for 10 minutes at 4°C, and take the supernatant. the

④ Transfer the supernatant to three 1.5ml centrifuge tubes, add chloroform, shake for 15 seconds, and place at room temperature for 5 minutes. The amount of chloroform added is 0.6ml chloroform for every 3ml TRizol used. the

⑤Centrifuge at 12000g for 15min at 4°C, take 500ul of the supernatant and transfer it to a new 2ml centrifuge tube. the

⑥ Add an equal volume of isopropanol to the supernatant (precipitate RNA in the aqueous phase), mix well and place at room temperature for 10 minutes. the

⑦ Centrifuge at 12000g for 10min at 4°C. A gelatinous precipitate appears on the side of the tube and at the bottom of the tube, discard the supernatant (if the quality of the precipitate is not good, on the basis of this precipitate, add TRizol again for extraction). the

⑧ Wash the RNA pellet with 75% ethanol (pre-cooled on ice, prepared with DEPC water). Add at least 3ml of 75% ethanol for every 3ml of TRizol used, shake a few times, centrifuge at no more than 7500g for 5min, and discard the supernatant. the

⑨ Dry at room temperature for 10 minutes, add 40ul of DEPC water, pipette several times with a pipette tip, and place at 55°C for 10 minutes to dissolve the RNA. the

⑩Determination of Total RNA concentration: Use NanoDrop 2000 to detect the concentration and A260/A280 value of the extracted RNA. the

3. cDNA synthesis

① Dilute the above-mentioned qualified RNA 40 times (3ulRNA+117ulDEPC water).

② Heat denature the RNA at 65°C for 5 minutes, and immediately place it on ice to cool. the

③ Use the reverse transcription kit to complete the synthesis of cDNA, and configure the following reaction solution (50ul system):

Total RNA, 0.6ul (note that the amount of Total RNA used is 500-1000ng); 5*RTbuffer, 10ul; Primer mix, 2.5ul; RT Enzyme Mix, 2.5ul; RNase free dH ₂ O was added to a total reaction volume of 50ul.

④ Reaction conditions: 37°C for 15 minutes (reverse transcription reaction)

98°C 5min (enzyme inactivation reaction)

⑤ Take it out and put it on ice for later use, or store it at -20°C.

⑥ Determination of cDNA concentration: use NanoDrop 2000 to measure the concentration of cDNA and the value of A260/A280. the

, primer design

We carried out qRT-PC screening and verification on the bee storage protein (hex71) gene, and designed primers for the gene using primer5.0 and Primer3 Plus software, and Shanghai Sangon Bioengineering Co., Ltd. completed the primer synthesis. A total of 2 pairs of primers were designed and synthesized, including 1 verification gene and 1 reference gene GAPDH (control). The most important principle for designing and selecting primers is to first ensure the specificity of the primers and accurately amplify the target product; secondly, the size of the amplified product is 80-250bp, because the amplified fragments in this range are more suitable for qRT- In the PCR method, too long amplified fragments will reduce the amplification efficiency and easily lead to non-specific reactions, and too short amplified fragments will also affect the non-specific amplification of the product and affect the accuracy of quantification; finally, the GC content of the primers used is between 40 -60%, and the annealing temperature (Tm value) should be kept consistent, which is conducive to the setting of the overall reaction conditions. The primer sequence results are shown in Table 1.

the

Table 1 The primer sequence of the target gene

5. qRT-PCR reaction system and conditions

Dilute the bee head cDNA obtained in the above step 3 by 6 times volume (5ul stock solution + 25ul DEPC water), and set aside. And prepare the 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:

Pre-denaturation 95°C, 30s

PCR reaction (40 cycles)

95℃, 5s

61℃, 31s

  Melting curve   55°C, 20s

6. Identify the yield performance of royal jelly

GAPDH, which expresses very stably in the head of bees, was selected as an internal reference gene, and the expression level of the target gene was calculated by the 2 ^-ΔΔct method. The relative expression of the target gene=2 ^-△△Ct , △△Ct = (Ct _target -Ct _GAPDH ) _treatment - (Ct _target -Ct _GAPDH ) _control , and the group of bees with the lowest royal jelly production was selected as the control group, The other 13 colonies of bees represented the treatment.

The target gene was repeated three times in each qRT-PCR reaction of each bee colony sample, and the 2- ^△△Ct average value of the three reactions was taken as the final expression level of the gene in the bee colony sample, and the final expression level of the bee hex71 gene The higher the expression level, the better the yield performance of royal jelly; finally, the unpaired sample mean t test method in SPSS13.0 software was used for statistics on the differential expression of genes between the bee samples of the high and low royal jelly yield groups (5 bee colonies in each group) Analysis, when 0.01<P<0.05, the difference is significant; when P<0.01, the difference is extremely significant.

Example 2, the expression abundance of hex71 gene in bees with high yield of royal jelly is significantly higher than that in bees with low yield of royal jelly

By counting the royal jelly production of 30 colonies of bees in the apiary five times in a row, 5 colonies with high royal jelly production and 5 colonies with low royal jelly production were finally screened. The results showed that the average yield of royal jelly in five colonies and five times of low-yield royal jelly group was 64.32g, and the average yield of royal jelly in five groups of five times in high-yield royal jelly group reached 115.64g. The results showed that F _1,40 =76.3935, P=0.0001, that is, there was a significant difference in the yield of royal jelly between high and low royal jelly yield groups.

The expression abundance of honeybee hex71 gene was detected by qRT-PCR technology. The expression abundance of the hex71 gene in the five colonies of bees in the high-yield royal jelly group was 11.6625±5.459, and the expression abundance of the hex71 gene in the five colonies of the bees in the low-yield royal jelly group was 2.797±1.6883, which was found by T-test analysis , the expression of honeybee hex71 gene was significantly different between high and low royal jelly production groups (P=0.0008). the

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Claims (6)

1. A method utilizing honeybee hex71 gene fluorescent quantitative PCR technology to identify royal jelly yield performance, characterized in that the identification method is as follows: (1) Sampling of bees: 30 nurturing bees were collected from each colony under investigation; the nurturing bees refer to worker bees that feed larvae in the bee colony, that is, worker bees that are 6-12 days old; (2) RNA extraction and cDNA synthesis from the bee head: extract the RNA from the head of the breeding bee collected, and reverse transcribe to obtain cDNA; (3) Primer design: The designed and synthesized primer sequences are as follows: Primer-F: 5' - agtatggctggttggcttattg - 3' Primer-R: 5' - cgatgttggcttctatgttcc-3'; (4) Fluorescence quantitative PCR detection: use the diluted bee head cDNA as a template, and use Primer-F and Primer-R as primers to perform fluorescence quantitative PCR; select GAPDH from the bee head as an internal reference gene, and use 2 ^{- △△ The ct} method is used to calculate the expression level of the bee hex71 gene; it is repeated several times in each qRT-PCR reaction of each bee colony sample, and the average value of multiple repetitions is taken as the final expression level of the honeybee hex71 gene, and the higher the final expression level , the better the royal jelly production performance of the bee colony; the 2 ^-△△ct is the relative expression level of the bee hex71 gene, where △△Ct = (Ct _target -Ct _GAPDH ) _treatment - (Ct _target -Ct _GAPDH ) _control . 2. a kind of method utilizing honeybee hex71 gene fluorescent quantitative PCR technology to discriminate royal jelly yield performance according to claim 1, it is characterized in that the reaction system of described fluorescent quantitative PCR is: total volume is 20ul, contains SYBR GreenⅠ 10ul, Rox 0.4ul, Primer-F 0.4ul, Primer-R 0.4ul, cDNA 2ul, DEPC water to make up to 20ul. 3. a kind of method utilizing honeybee hex71 gene fluorescent quantitative PCR technology to discriminate royal jelly yield performance according to claim 1, it is characterized in that the reaction condition of described fluorescent quantitative PCR is as follows: pre-denaturation 95 ℃, 30s, PCR reaction 40 cycles , 95℃, 5s, 61℃, 31s, melting curve 55℃, 20s. 4. a kind of method utilizing honeybee hex71 gene fluorescent quantitative PCR technology to discriminate royal jelly output performance according to claim 1, it is characterized in that described bee head RNA is extracted and cDNA is synthesized, adopts TRizol method to extract RNA, adopts reverse transcription The kit completes the synthesis of cDNA. 5. a kind of method utilizing honeybee hex71 gene fluorescent quantitative PCR technology to discriminate royal jelly yield performance according to claim 1, it is characterized in that the honeybee head cDNA after described dilution, promptly dilute honeybee head cDNA with DEPC water 6. double the volume. 6. a kind of method utilizing honeybee hex71 gene fluorescent quantitative PCR technology to differentiate royal jelly yield performance according to claim 1, it is characterized in that described in each qRT-PCR reaction of each bee colony sample all repeats multiple times, The number of repetitions is 3 times.