CN113265419A - Rapid verification method for apple fruit size gene function - Google Patents
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Abstract
A method for quickly verifying the gene function of apple fruit includes cloning candidate gene, silencing candidate gene or constructing overexpression vector, infecting candidate gene, analyzing phenotype and gene identification of apple fruit sample, and verifying and analyzing candidate gene, and features that instantaneous function verifying technology is used to directly infect candidate gene in apple fruit in injection mode at specific development period of target sample, and Agrobacterium directly infects target part at key development period of fruit to quickly express and verify gene function, so shortening the gene function verifying period from 7-9 years to 1 year, meeting the requirement of high efficiency in fruit gene function verification in production field of perennial crops such as apple, the verification process is simplified, the verification cost is low, and the verification efficiency is improved.
Description
Technical Field
The invention relates to a method for verifying gene functions of apples, in particular to a method for quickly verifying gene functions of apple fruits.
Background
China is the origin center of world apple cultivation, the apple germplasm resources are rich, the cultivation region is wide, the cultivation area and the yield are always the first in the world for years, and the annual yield accounts for 40 percent of the total annual yield in the world. The genetic and molecular mechanism of the apple germplasm is analyzed, the size development of the fruits is regulated and controlled by using a molecular biological technical method, the yield of the apples is improved, and the gene functions of different varieties of apples need to be extensively and deeply researched. However, since apples are perennial crops, the seedling growth juvenile period is long, and fruits are visible in 6-8 years after sowing, at least 7-9 years of cultivation period is needed for obtaining the expression characteristics of gene functions and verifying the gene functions through the apples, and the long test period seriously restricts the application of the apple character gene functions. At present, the gene function verification in the apple mainly aims at the leaf or plant and other nutritive organs, and the verification of flower, fruit and other reproductive organs is rarely reported. The traditional transgenic function verification method comprises the steps of cloning candidate genes, transforming agrobacterium tumefaciens, co-culturing bacterial liquid and leaves, screening stable transgenic plants by a resistance culture medium, verifying transgenic effect by qRT-PCR and the like, and has the disadvantages of complex operation process, higher verification cost and longer test period; moreover, the apple varieties have the problems that a stable transgenic system is difficult to obtain and plants are difficult to obtain, the gene function verification of the apple fruit size and character and the accurate gene function acquisition have adverse effects, great troubles are brought to the scientific research of the gene function verification of the apples, and the gene function verification becomes a limiting factor for restricting the scientific research of the apples. Therefore, it is necessary to research and develop a method for verifying the size and character of apple fruits, which can quickly verify the function of candidate genes by the transient gene expression characteristics, simplify the verification process, shorten the verification period, reduce the verification cost and improve the verification efficiency.
Disclosure of Invention
The invention aims to provide a rapid verification method for the gene function of apple fruit size, which shortens the verification period and improves the verification efficiency.
A rapid verification method for apple fruit size gene function comprises the following steps:
step 1, cloning of candidate Gene
Designing a full-length primer of a candidate gene to be verified, amplifying the full length of a sequence, connecting a T vector for sequencing, comparing the obtained candidate gene sequence with a reference sequence, determining the actual sequence of the candidate gene, and preliminarily analyzing the possible functions of the candidate gene, the correlation between the candidate gene and the fruit size difference, and negative or positive expression and regulation characteristics.
Step 2, construction of silencing or over-expression vector of candidate gene
Determining a verification test vector, selecting and constructing a silencing or overexpression vector of the cloned candidate gene in the step 1 according to the difference of the negative or positive expression and regulation characteristics of the candidate gene, then transforming the constructed silencing or overexpression vector of the candidate gene into agrobacterium, preparing agrobacterium verification bacterial liquid carrying the candidate gene through suspension treatment, and meanwhile reserving agrobacterium no-load bacterial liquid not carrying the candidate gene for later use. Wherein:
when the verification test vector is selected as a small fruit sample, the candidate gene is negative expression and regulation characteristics, or the verification test vector is selected as a large fruit sample, and the candidate gene is positive expression and regulation characteristics, constructing a silencing vector of the candidate gene;
and when the verification test vector is selected as a small fruit sample and the candidate gene is positively expressed and has a regulating characteristic, or the verification test vector is selected as a large fruit sample and the candidate gene is negatively expressed and has a regulating characteristic, constructing an overexpression vector of the candidate gene.
Step 3, infection of candidate genes
And (2) aiming at the selected verification test carrier, in the cell enlargement initial stage of apple fruit growth, respectively selecting apple fruit samples with the same quantity and the similar size to divide the apple fruit samples into a test group and a control group, respectively injecting the agrobacterium verification bacterial liquid prepared in the step (2) and the reserved agrobacterium no-load bacterial liquid into the apple fruit samples of the test group and the control group in an external injection infection mode, wherein the injection operation needs to be kept stable at a constant speed, and when liquid seeps from the surface around the fruit, the agrobacterium verification bacterial liquid and the agrobacterium no-load bacterial liquid are uniformly distributed in the apple fruit samples, stopping injection and completing the infection operation.
After the infection operation is completed, the apple fruits grow for 15-30 days, and the apple fruit samples of the test group and the apple fruit samples of the control group are picked in groups for later use.
Step 4, phenotype analysis of apple fruit sample
Taking the apple fruit samples of the test group and the control group picked in the step 3, respectively measuring the transverse diameter and the longitudinal diameter of each apple fruit sample, weighing the monomer weight, performing statistical analysis on the obtained data, and respectively calculating to obtain the average transverse diameter D of the test group of the apple fruit samples1Average longitudinal diameter H of test group1And test group average monomer weight W1Value, and control group mean transverse diameter D0Average longitudinal diameter H of control group0And control group average monomer weight W0The value is obtained.
Step 5, identifying genes of apple fruit samples
Rapidly freezing the apple fruits of the test group and the control group picked in the step 3 by using liquid nitrogen to prepare frozen samples, extracting the total RNA of the apple fruits and respectively converting the total RNA into cDNA step by step, then detecting the expression quantity of the candidate gene in the cDNA of the apple fruit sample, respectively calculating the average expression quantity N of the candidate gene of the test group of the apple fruit sample after statistically analyzing the detection data1And the average expression level N of the candidate genes in the control group0The value is obtained.
Step 6, verification analysis of candidate genes
Respectively testing the average transverse diameter D of the apple fruit sample test group obtained in the step 41Average longitudinal diameter H of test group1And test group average monomer weight W1The value is equal to the average transverse diameter D of the control group obtained in the same step0Average longitudinal diameter H of control group0And control group average monomer weight W0Carrying out corresponding comparison on the values, and carrying out average expression quantity N of candidate genes of the test group of the apple fruit sample obtained in the step 51The value is equal to the average expression quantity N of candidate genes in the test group obtained in the same step0The values were compared.
When small fruit samples are selected as verification test vectors to verify negative expression and regulatory genes, the average transverse diameter D of the test group1The average longitudinal diameter H of the test group1And the average monomer weight W of the test group1The values are respectively larger than the average transverse diameter D of the control group0The average longitudinal diameter H of the control group0And the average monomer weight W of the control group0Value, and the average expression level N of the candidate genes in the test group1The value is less than the average expression quantity N of the candidate genes in the control group0If so, judging that the candidate gene has the function of regulating the fruit size, otherwise, judging that the candidate gene does not have the function of regulating the fruit size.
When a large fruit sample is selected as a verification test carrier to verify the negative expression and regulatory gene, the average transverse diameter D of the test group1The average longitudinal diameter H of the test group1And the average monomer weight W of the test group1Values less than the control mean transverse diameter D0The average longitudinal diameter H of the control group0And the average monomer weight W of the control group0Value, and the average expression level N of the candidate genes in the test group1The value is larger than the average expression quantity N of the candidate genes in the control group0If so, judging that the candidate gene has the function of regulating the fruit size, otherwise, judging that the candidate gene does not have the function of regulating the fruit size.
Selecting small fruit sample as verification test carrier to verify positive expressionAnd when regulating genes, the mean transverse diameter D of the test group1The average longitudinal diameter H of the test group1And the average monomer weight W of the test group1The values are respectively larger than the average transverse diameter D of the control group0The average longitudinal diameter H of the control group0And the average monomer weight W of the control group0Value, and the average expression level N of the candidate genes in the test group1The value is larger than the average expression quantity N of the candidate genes in the control group0If so, judging that the candidate gene has the function of regulating the fruit size, otherwise, judging that the candidate gene does not have the function of regulating the fruit size.
When a large fruit sample is selected as a verification test carrier to verify the positive expression and regulatory gene, the average transverse diameter D of the test group1The average longitudinal diameter H of the test group1And the average monomer weight W of the test group1Values less than the control mean transverse diameter D0The average longitudinal diameter H of the control group0And the average monomer weight W of the control group0Value, and the average expression level N of the candidate genes in the test group1The value is less than the average expression quantity N of the candidate genes in the control group0If so, judging that the candidate gene has the function of regulating the fruit size, otherwise, judging that the candidate gene does not have the function of regulating the fruit size.
The rapid verification method for the gene function of the apple fruit size comprises the following steps:
in the step 1, preferably, 1-2 pairs of full-length primers of the candidate genes are designed at one time, and primers with high amplification efficiency are selected from the primers for amplification to screen genes;
in step 2, after the silent vector is transformed into agrobacterium, preferably, a universal primer is used for PCR amplification, a positive strain is screened, and agrobacterium liquid of the candidate gene is cultured by shaking.
In the step 3, the period of the external injection infection on the apple fruit sample is preferably 30-36 days after the full bloom.
In step 3, the trunk part of the apple fruit with moderate hardness and far distance from the fruit core is preferably selected as an injection part, and the hormone level of the apple fruit is not changed by damaging the key structure of the fruit by taking the part as an injection area, so that the hormone level has no influence on the verification result.
In the step 3, preferably, a 1 ml syringe injection needle is used for injecting the apple fruits, the depth of the injection needle inserted into the apple fruits is 0.7-1 cm, and the injection speed is kept at 0.05-0.15 ml/s for uniform injection.
The method has the beneficial effect of providing a rapid verification method for the gene function of the apple fruit size. The method adopts an instantaneous function verification technology, and in a specific development period of a target sample apple fruit, candidate genes are directly infected into the apple fruit in an injection mode, and agrobacterium tumefaciens directly infects a target part in a key development period of the fruit, so that the aim of quickly expressing and verifying the gene function is achieved, the problems of long childbearing period of the apple fruit, long fruit gene function verification period, complex operation process and high verification cost in the prior art are solved, the apple fruit gene function verification period is shortened to less than 1 year from 7-9 years, the requirements of the current scientific research and production fields of perennial crops such as apples on the quick and high efficiency of fruit gene function verification are met, meanwhile, the verification process can be simplified, the verification cost is low, and the verification efficiency is improved.
Drawings
FIG. 1 is a graph showing the fruit phenotype of the test and control samples after the "Longfeng" and "DaguoLongfeng" apples are injected and infested for growth for 25 days.
FIG. 2 is a graph showing the comparison of the expression levels of MdAux/IAAa genes in the test group and the control group after 25 days of infection with "Longfeng" and "DaguoLongfeng" apples by injection.
Detailed Description
The claimed technical solution is further described below with reference to specific embodiments.
Example 1
Aiming at the difference of fruits between a ' Longfeng ' apple fixedly planted in Ning ' city of Heilongjiang province and a ' Daguofeng ' apple cultivated by the ' Longfeng ' big fruit type bud mutation, the ' Longfeng ' apple is taken as a verification test carrier to perform function instant verification on the genes related to the size of the apple fruits possibly existing between the ' Longfeng ' apple and the ' Daguofeng ' apple, and the method specifically comprises the following steps:
step 1, determination of candidate genes
Respectively extracting total RNA in the fruits of the Longfeng and the Longfeng by using a CTAB method, wherein the RNA extraction ensures that the semi-quantitative detection of 28S and 18S is clear and visible, and the thickness of the 28S band is 2 times of that of the 18S band, then respectively converting the RNA into cDNA by using a reverse kit through two steps, respectively carrying out PCR detection on the cDNA by using reference genes MdActin, MdActin-F and MdActin-R as primers, ensuring that the concentrations of the cDNA are consistent, and showing that the difference expression exists between the MdAux/IAAa genes through transcription group sequencing, further designing an expression primer of the MdAux/IAA genes by using a primer premier 6, and preliminarily judging that the MdAux/IAAa genes can be negative expression with the function of controlling the fruit size and regulating the apple fruit size through qRT-PCR identification, and determining the MdAux/IAAa gene as a verification candidate gene.
Step 2, cloning of candidate Gene
The method comprises the steps of designing a candidate gene MdAux/IAAa full-length primer by using primer premier 6, amplifying the full length of a sequence by using a conventional PCR method, connecting a T vector to sequencing by committee bioengineering (Shanghai) corporation, comparing the obtained sequence with the MdAux/IAAa in a reference genome by using Sequencher software, determining the gene sequence of the candidate gene MdAux/IAAa in the step 1, finding that the MdAux/IAAa gene is close to the genetic relationship related to the size of an organ, and presuming that the candidate gene MdAux/IAAa is possibly related to the size difference of the fruit.
Step 3, construction of candidate gene MdAux/IAAa silencing vector
The candidate gene MdAux/IAAa is a negative expression and regulation gene, the MdAux/IAAa gene cloned in the step 2 is connected with a pRI101 (35S promoter drive) vector to construct a silencing vector of the candidate gene MdAux/IAAa, further, the silencing vector is transformed into agrobacterium, PCR amplification is carried out by using a universal primer, a positive strain is screened, agrobacterium tumefaciens liquid of the candidate gene MdAux/IAAa is cultured by shaking bacteria, agrobacterium validation bacterial liquid carrying the MdAux/IAAa gene is prepared by suspension treatment, and meanwhile, agrobacterium tumefaciens no-load bacterial liquid which does not carry the MdAux/IAA gene is reserved for standby.
Step 4, infection of candidate gene MdAux/IAAa
Respectively selecting 10 apple fruit samples with similar sizes to form a test group and a control group 30 days after the growth and blooming of the apple of the verification test carrier Longfeng, injecting the agrobacterium verification bacterial liquid prepared in the step 3 into 10 apple fruit samples of the test group by adopting an external injection mode, injecting the agrobacterium no-load bacterial liquid reserved in the step 3 into 10 apple fruit samples of the control group, using a 1 ml injector for injection operation, wherein the injection part is the trunk part of the apple fruit sample, a needle is vertically inserted into the apple, the insertion depth is controlled to be 0.8 cm, the uniform injection speed of 0.1 ml/second is kept, so as to ensure the bacterial liquid permeation effect and prevent the damage to the pulp cell tissues, and when the liquid exudation is observed on the skin around the apple fruit in the injection process, the agrobacterium verification bacterial liquid and the agrobacterium no-load bacterial liquid are uniformly distributed in the apple fruit samples, stopping injection and finishing infection operation.
After the infection operation is finished, the apple fruits grow for 25 days, and the apple fruit samples of the test group and the apple fruit samples of the control group are picked in groups for later use.
Step 5, phenotype analysis of apple fruit sample
Taking the apple fruit samples of the test group and the control group picked in the step 4, sequentially measuring the transverse diameter and the longitudinal diameter of each apple fruit sample by using a digital display vernier caliper, weighing the monomer weight of the apple fruit samples by using an electronic balance, and performing statistical analysis on the two groups of acquired data by using SPSS software, wherein the result shows that P is less than 0.01, the data difference has significant statistical significance, as shown in figure 1, through data statistical calculation, the average transverse diameter D of the test group of the apple fruit samples143.8 mm, test group average longitudinal diameter H132.2 mm, average monomer weight W of test group132.8 g, mean transverse diameter D of control group038.2 mm, average longitudinal diameter H of control group0Is 31.2 mmAnd control group average monomer weight W0It was 27.3 g.
Step 6, identifying genes of apple fruit samples
Rapidly freezing the apple fruit slices of the test group and the control group picked in the step 4 by using liquid nitrogen to prepare a frozen sample, extracting the total RNA of the apple fruit by using a CTAB method, converting the total RNA into cDNA step by using a reverse transcription kit, detecting the expression quantity of the candidate gene MdAux/IAAa in the cDNA of the apple fruit sample by using qRT-PCR (quantitative reverse transcription-polymerase chain reaction), statistically analyzing the two groups of obtained data by using SPSS (Spanish space switch) software, and respectively calculating the average expression quantity N of the candidate gene of the test group of the apple fruit sample, wherein the result shows that P < 0.01, the data difference has significant statistical significance, as shown in figure 2, and the average expression quantity N of the candidate gene of the test group of the apple fruit sample is obtained1Is 0.45X 10-4And the average expression level N of candidate genes in the control group0Is 1.36X 10-4。
Step 7, verification analysis of candidate genes
As the 'Longfeng' apple is a small fruit sample verification test carrier and the candidate gene MdAux/IAAa is a negative expression and regulation gene, the comparison of the experimental group obtained in the step 5 and the control group shows that the apple fruit sample phenotype analysis data shows that the average transverse diameter D of the experimental group1Larger than the average transverse diameter D of the control group0The average longitudinal diameter H of the test group1Greater than the mean longitudinal diameter H of the control group0Average monomer weight W of the test group1Greater than the average monomer weight W of the control group0Meanwhile, comparing the gene expression quantity data of the apple fruit sample of the experimental group and the control group obtained in the step 6, the average expression quantity N of the candidate genes of the experimental group can be seen1Less than the average expression level N of the candidate genes in the control group0And based on the action relation and change rule between the differential expression of the candidate gene MdAux/IAAa and the sample size of the apple fruit, the candidate gene MdAux/IAAa can be judged to have the function of regulating the size of the apple fruit.
Example 2
Aiming at the difference of fruits between a ' Longfeng ' apple fixedly planted in Ning ' city of Heilongjiang province and a ' DaguoLongfeng ' apple cultivated by the ' Longfeng ' big fruit type bud mutation, the ' DaguoLongfeng ' apple is taken as a verification test carrier to perform function instant verification on the related genes of the size of the apple fruits possibly existing between the ' Longfeng ' apple and the big fruit type bud mutation, and the method specifically comprises the following steps:
step 1 and step 2 were carried out by exactly the same procedure as in example 1 to determine and clone the candidate gene MdAux/IAAa, respectively.
Step 3, construction of candidate gene MdAux/IAAa overexpression vector
The candidate gene MdAux/IAAa is a negative expression and regulation gene, the MdAux/IAAa gene cloned in the step 2 is connected with a pRI101 (35S promoter drive) vector to construct an over-expression vector of the candidate gene MdAux/IAAa, the over-expression vector is further transformed into agrobacterium, PCR amplification is carried out by using a universal primer, a positive strain is screened, agrobacterium liquid of the candidate gene MdAux/IAAa is cultured by shaking bacteria, agrobacterium validation bacterial liquid carrying the MdAux/IAAa gene is prepared by suspension treatment, and meanwhile, agrobacterium empty bacterial liquid which does not carry the MdAux/IAAa gene is reserved for standby.
Step 4, infection of candidate gene MdAux/IAAa
Respectively selecting 10 apple fruit samples with the sizes corresponding to the approximate sizes to form a test group and a control group 30 days after the growth and blooming of the verification test carrier 'Daguofeng' apple, injecting the agrobacterium verification bacterial liquid prepared in the step 3 into the 10 apple fruit samples in the test group by adopting an external injection mode, injecting the agrobacterium no-load bacterial liquid reserved in the step 3 into the 10 apple fruit samples in the control group, injecting the agrobacterium no-load bacterial liquid by using a 1 ml injector, wherein the injection part is the trunk part of the apple fruit sample, a needle is vertically inserted into the apple, the insertion depth is controlled to be 0.8 cm, the constant injection speed of 0.1 ml/sec is kept, so as to ensure the bacterial liquid permeation effect, prevent the pulp cell tissue from being damaged, and when the liquid exudation is observed on the surface around the apple fruit in the injection process, the agrobacterium verification bacterial liquid and the agrobacterium no-load bacterial liquid are uniformly distributed in the apple fruit samples, stopping injection and finishing infection operation.
After the infection operation is finished, the apple fruits grow for 25 days, and the apple fruit samples of the test group and the apple fruit samples of the control group are picked in groups for later use.
Step 5, phenotype analysis of apple fruit sample
The apple fruit samples of the test group and the control group were phenotypically analyzed by the same operation method as in example 1, and the average transverse diameter D of the test group of the apple fruit samples was statistically calculated as shown in FIG. 1137.5 mm, test group average longitudinal diameter H128.9 mm, average test group monomer weight W123.8 g, mean transverse diameter D of control group051.0 mm, mean longitudinal diameter H of control group034.9 mm and the average monomer weight W of the control group048.3 grams.
Step 6, identifying genes of apple fruit samples
Performing gene identification on the apple fruit samples of the test group and the control group by the same operation method as in example 1, and calculating the average expression level N of candidate genes of the test group of the apple fruit samples respectively as shown in FIG. 21Is 1.92 multiplied by 10-4And the average expression level N of candidate genes in the control group0Is 0.92X 10-4。
Step 7, verification analysis of candidate genes
As the 'Daguofeng' apple is a large fruit sample verification test carrier and the candidate gene MdAux/IAAa is a negative expression and regulation gene, the phenotypic analysis data of the apple fruit samples of the experimental group and the control group obtained in the comparison step 5 can be seen, and the average transverse diameter D of the experimental group is1Smaller than the average transverse diameter D of the control group0The average longitudinal diameter H of the test group1Less than the mean longitudinal diameter H of the control group0Average monomer weight W of the test group1Less than the average monomer weight W of the control group0Meanwhile, comparing the gene expression quantity data of the apple fruit sample of the experimental group and the control group obtained in the step 6, the average expression quantity N of the candidate genes of the experimental group can be seen1Is larger than the average expression quantity N of the candidate genes in the control group0Based on the action relationship and change rule between the candidate gene MdAux/IAAa differential expression and the sample size of the apple fruit, the method can judgeThe candidate gene MdAux/IAAa has the function of regulating the size of apple fruits.
Example 3
A 'Longfeng' apple is taken as a verification test carrier, and a comparative test of infection effects of candidate genes MdAux/IAAa in different apple growth periods is carried out.
In the initial period of cell enlargement of apple fruit growth, 30 days after full bloom, 51 days after full bloom, 72 days after full bloom and 93 days after full bloom are respectively selected as injection test periods, 10 apple fruit samples are respectively selected in each injection test period, the selected apple fruit samples are respectively subjected to infection injection by adopting the same injection mode as the step 4 in the example 1, the integrity and the distribution condition of agrobacterium of the apple fruit samples are detected after 2 hours of injection, and the statistical conditions of the detected data are shown in table 1.
Test results show that when agrobacterium is injected 51, 72 and 93 days after the full-bloom period, the quantity of the agrobacterium liquid which can be injected into each apple fruit sample is small on one hand due to high fruit hardness, and infiltration and effective diffusion of infection liquid are not easy on the other hand, and data show that the agrobacterium liquid is injected and infected in the infection period, the average infection proportion of the sample can only reach 40% of the fruit volume at most, and the requirements of a gene function verification test can not be met; moreover, because the fruit is brittle, the fruit cracking phenomenon can be caused by the change of internal stress after a small amount of injection of the agrobacterium liquid, the further growth of the fruit is influenced, and the gene function verification test cannot be continued.
Meanwhile, the experiment of the embodiment fully proves that the hardness of the fruits is proper relative to the hardness in the early stage and the late stage and the brittleness is proper, the mechanical resistance of the agrobacterium liquid injection and infection in the period is proper, the fruit cracking tendency is low, the permeability of the liquid is good, and the liquid can be uniformly distributed and permeate into the whole fruits under the condition of not moving a needle head, in the experiment of the embodiment, the cracking condition of the fruit sample does not occur in the apple fruit sample injected and infected in the period, the average infection rate of the fruit sample also reaches 100%, and the 30 days after full bloom is the optimal period for the apple fruit sample injection and infected, and is also a key technical link for realizing the technical scheme of the invention.
Claims (6)
1. A rapid verification method for apple fruit size gene function is characterized by comprising the following steps:
step 1, cloning of candidate Gene
Designing a full-length primer of a candidate gene to be verified, amplifying the full length of a sequence, connecting a T vector for sequencing, comparing the obtained candidate gene sequence with a reference sequence, determining the actual sequence of the candidate gene, and preliminarily analyzing the possible functions of the candidate gene, the correlation between the candidate gene and the fruit size difference, and negative or positive expression and regulation characteristics;
step 2, construction of silencing or over-expression vector of candidate gene
Determining a verification test vector, selecting and constructing a silencing or overexpression vector of the cloned candidate gene in the step 1 according to the difference of the negative or positive expression and regulation characteristics of the candidate gene, then transforming the constructed silencing or overexpression vector of the candidate gene into agrobacterium, preparing agrobacterium verification bacterial liquid carrying the candidate gene through suspension treatment, and meanwhile reserving agrobacterium no-load bacterial liquid not carrying the candidate gene for later use; wherein:
when the verification test vector is selected as a small fruit sample, the candidate gene is negative expression and regulation characteristic, or the verification test vector is selected as a large fruit sample, and the candidate gene is positive expression and regulation characteristic, constructing a silencing vector of the candidate gene;
when the verification test vector is selected as a small fruit sample, the candidate gene is characterized by positive expression and regulation characteristics, or the verification test vector is selected as a large fruit sample, and the candidate gene is characterized by negative expression and regulation characteristics, constructing an overexpression vector of the candidate gene;
step 3, infection of candidate genes
Aiming at the selected verification test carrier, in the cell enlargement initial period of apple fruit growth, respectively selecting apple fruit samples with the same quantity and the similar size to divide the apple fruit samples into a test group and a control group, respectively injecting the agrobacterium verification bacterial liquid prepared in the step 2 and the reserved agrobacterium no-load bacterial liquid into the apple fruit samples of the test group and the control group by adopting an external injection infection mode, wherein the injection operation needs to be kept stable at a constant speed, and when liquid seeps from the surface around the fruit, the agrobacterium verification bacterial liquid and the agrobacterium no-load bacterial liquid are uniformly distributed in the apple fruit samples, stopping injection and completing the infection operation;
after the infection operation is completed, growing for 15-30 days, and picking apple fruit samples of the test group and the control group in groups for later use;
step 4, phenotype analysis of apple fruit sample
Taking the apple fruit samples of the test group and the control group picked in the step 3, respectively measuring the transverse diameter and the longitudinal diameter of each apple fruit sample, weighing the monomer weight, performing statistical analysis on the obtained data, and respectively calculating to obtain the average transverse diameter D of the test group of the apple fruit samples1Average longitudinal diameter H of test group1And test group average monomer weight W1Value, and control group mean transverse diameter D0Average longitudinal diameter H of control group0And control group average monomer weight W0A value;
step 5, identifying genes of apple fruit samples
Rapidly freezing the apple fruits of the test group and the control group picked in the step 3 by using liquid nitrogen to prepare frozen samples, extracting the total RNA of the apple fruits and respectively converting the total RNA into cDNA step by step, then detecting the expression quantity of the candidate gene in the cDNA of the apple fruit sample, respectively calculating the average expression quantity N of the candidate gene of the test group of the apple fruit sample after statistically analyzing the detection data1And the average expression level N of the candidate genes in the control group0A value;
step 6, verification analysis of candidate genes
Respectively testing the average transverse diameter D of the apple fruit sample test group obtained in the step 41Average longitudinal diameter H of test group1And test group average monomer weight W1The value is equal to the average transverse diameter D of the control group obtained in the same step0Average longitudinal diameter H of control group0And control group average monomer weight W0Carrying out corresponding comparison on the values, and carrying out average expression quantity N of candidate genes of the test group of the apple fruit sample obtained in the step 51The value is equal to the average expression quantity N of candidate genes in the test group obtained in the same step0Comparing the values;
when small fruit samples are selected as verification test vectors to verify negative expression and regulatory genes, the average transverse diameter D of the test group1The average longitudinal diameter H of the test group1And the average monomer weight W of the test group1The values are respectively larger than the average transverse diameter D of the control group0The average longitudinal diameter H of the control group0And the average monomer weight W of the control group0Value, and the average expression level N of the candidate genes in the test group1The value is less than the average expression quantity N of the candidate genes in the control group0If so, judging that the candidate gene has the function of regulating the size of the fruit, otherwise, judging that the candidate gene does not have the function of regulating the size of the fruit;
when a large fruit sample is selected as a verification test carrier to verify the negative expression and regulatory gene, the average transverse diameter D of the test group1The average longitudinal diameter H of the test group1And the average monomer weight W of the test group1Values less than the control mean transverse diameter D0The average longitudinal diameter H of the control group0And the average monomer weight W of the control group0Value, and the average expression level N of the candidate genes in the test group1The value is larger than the average expression quantity N of the candidate genes in the control group0If so, judging that the candidate gene has the function of regulating the size of the fruit, otherwise, judging that the candidate gene does not have the function of regulating the size of the fruit;
when selecting small fruit samples as verification test vectors to verify the positive expression and regulatory genes, the average transverse diameter D of the test group1The average longitudinal diameter H of the test group1And the average monomer weight W of the test group1The values are respectively larger than the average transverse diameter D of the control group0The average longitudinal diameter H of the control group0And the average monomer weight W of the control group0Value, and the average expression level N of the candidate genes in the test group1The value is larger than the average expression quantity N of the candidate genes in the control group0If so, judging that the candidate gene has the function of regulating the size of the fruit, otherwise, judging that the candidate gene does not have the function of regulating the size of the fruit;
when a large fruit sample is selected as a verification test carrier to verify the positive expression and regulatory gene, the average transverse diameter D of the test group1The average longitudinal diameter H of the test group1And the average monomer weight W of the test group1Values less than the control mean transverse diameter D0The average longitudinal diameter H of the control group0And the average monomer weight W of the control group0Value, and the average expression level N of the candidate genes in the test group1The value is less than the average expression quantity N of the candidate genes in the control group0If so, judging that the candidate gene has the function of regulating the fruit size, otherwise, judging that the candidate gene does not have the function of regulating the fruit size.
2. The method for rapidly verifying the gene function of apple fruit size according to claim 1, wherein: in the step 1, 1-2 pairs of full-length primers of the candidate genes are designed at one time, and primers with high amplification efficiency are selected from the primers for amplification, and genes are screened.
3. The method for rapidly verifying the gene function of apple fruit size according to claim 1, wherein: in step 2, after the silent or expression vector is transformed into agrobacterium, a universal primer is used for PCR amplification, a positive strain is screened, and agrobacterium liquid of the candidate gene is cultured by shaking.
4. The method for rapidly verifying the gene function of apple fruit size according to claim 1, wherein: in the step 3, the period of external injection infection on the apple fruit sample is 30-36 days after full bloom.
5. The method for rapidly verifying the gene function of apple fruit size according to claim 4, wherein: in step 3, the whole apple fruit is used as an injection site.
6. The method for rapidly verifying the function of the apple fruit size gene as claimed in claim 4 or 5, wherein: in the step 3, a 1 ml syringe needle is used, the depth of the needle inserted into the apple fruit is 0.7-1 cm, and the injection speed is kept at 0.05-0.15 ml/s for uniform injection.
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