CN117210490B - PCHR gene for regulating and controlling malus plant self-flower fructification and application thereof - Google Patents
PCHR gene for regulating and controlling malus plant self-flower fructification and application thereof Download PDFInfo
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Abstract
The invention relates to a PCHR gene for regulating and controlling malus plant self-flower fructification and application thereof. The invention discovers that inhibiting the expression of apple PCHR protein can regulate and control the self-fructification of apples, and is reflected in influencing the growth, extension and final fruit setting of pollen tubes in apple self-pollination flower columns. In practical application, the transcription level of the PCHR gene of the apples can be inhibited by means of transgenosis and the like, the self-pollination seed setting rate of the self-parthenocarpy variety is obviously improved, the self-parthenocarpy capability is obtained, the self-parthenocarpy of the apples is regulated, and the method has important application value in cultivating new varieties of the self-parthenocarpy apples.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a PCHR gene for regulating and controlling malus self-flower fructification and application thereof.
Background
Apples are important fruit tree species in China, and the planting area and the yield are all in the first place in the world. However, most cultivated apples have self-flower fecundity, show that self-pollination cannot be carried out, and the pollinating tree must be configured to rely on insect pollination or artificial pollination to ensure the yield, so that the difficulty and cost of apple production are greatly increased.
The subject group early uses the self-parthenocarpic apple variety 'Yue Shuai' as a male parent and uses the self-parthenocarpic apple variety 'Hanfu' as a female parent to create a hybrid offspring group separated from the self-parthenocarpic character. By means of genome-wide association analysis, the quantitative trait locus of 'Hanfu' self-flower fructification is located, and further analysis finds a key gene PCHR affecting self-flower fructification. The gene has the capability of combining free calcium ions and regulating the calcium ion steady state, and the low expression of the gene can promote the self-flower fructification of apples.
Calcium ions are important signal substances for various physiological reactions in cells, and play an important role in pollen germination and pollen tube growth. To date, it has been established that after germination of pollen from flowering plants on stigma, the tip of pollen tube establishes a high-to-low calcium ion concentration gradient. This difference in calcium ion concentration is critical to maintaining pollen tube integrity, extensibility and guidance. In addition, self-pollination of self-parthenocarpic plants often produces strong calcium oscillations that are used to initiate downstream reactions that regulate the growth of pollen tubes in the flower columns. However, it is not clear how calcium binding proteins specifically regulate calcium ion signals to affect apple self-flower fecundity.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a PCHR gene for regulating and controlling malus self-flower fructification and application thereof. The PCHR gene has the effect of regulating self-flower fructification of apples, and is reflected in influencing the growth of pollen tubes in self-pollination flower columns of apples, extension and final fruit setting. The invention promotes the self-pollination of malus plants incapable of self-anthesis by silencing PCHR gene, provides basis for analyzing the mechanism of self-anthesis of apples, and has practical significance for cultivating new varieties of self-anthesis of apples
In order to achieve the above purpose, the invention adopts the following technical scheme:
the application of the inhibitor of the coding gene of the PCHR protein of the malus plant in improving the self-flower setting capacity of the malus is characterized in that:
the amino acid sequence of the PCHR protein is shown as SEQ ID NO. 1:
SEQ ID NO.1：
MAFRVRNSSSLLSLVLLSLLAIASAKVFFEERFEDGWDKRWVKSDWKSDESLAGEWNYTSGKWNGDANDKGIQTSEDYRFYAISAEFPEFSNKDKTLVFQFSVKHEQKLDCGGGYIKLLSGDVDQKKFGGDTPYSIMFGPDICGYSTKKVHAILNYNNTNNLIKKDVPCETDQLTHVYTFILRPDATYSILIDNVEKQTGSLYSDWDLLPPKKIKDPEAKKPEDWDDKEYIPDPEDTKPEGYDDIPKEIVDPEAKKPEDWDDEEDGEWTAPTIPNPEYKGEWKPKKIKNPNFKGKWKAPLIDNPEFKDDPELYVYPNLKYVGIELWQVKSGTLFDNILITDEPEYAKQLAEETWGKQKDAEKAAFEEAERKQEEEAKDPVDSDAEEEDDADTDDAEDDSDAESKSDSTEESAEESEKHKC；
the inhibition factor is gRNA shown in SEQ ID NO. 2;
SEQ ID NO.2：
CACACCTCTGGCAAGTGGAATGGAGACTCTAATGACAAAGGTATCCAGACCAGCGAAGACTACAGGTTCCATGCCATTTCGGCTGAGTTCCCTGAATTTAGTAACAAGGGAAAAACCTTAGTATTCCAGTTCTCTGTTAAGCATGAGCAGAAGCTTGACTGTGGTGGTGGATACATGAAGTTGCTTAGTGGAGACGTTGACCAAAAGAAATTCGGTGGTGACACTCCCTACAGTATCATGTTTGGACCAGACATCTGTGGC；
the application is as follows: and carrying out transcriptional level inhibition on the coding gene of the apple PCHR protein by using the inhibiting factor of the coding gene of the apple PCHR protein so as to improve the self-flower setting capability of the apple.
A method for improving self-flower setting capability of apples is characterized in that gRNA with a nucleotide sequence shown as SEQ ID NO.2 is adopted to inhibit the transcription level of coding genes of apple PCHR proteins, so that the growth of pollen tubes for promoting self-pollination of apples is realized, and the fertilization success rate and the fruit setting rate of self-pollination are improved.
The application of the inhibitor of the coding gene of the PCHR protein of the malus plant in improving the self-flower setting capacity of the malus has the beneficial effects that:
the invention discovers that the apple PCHR protein participates in regulating and controlling the growth of pollen tubes in the self-pollination flower columns of apples, and extends and finally fruit bearing, and can obviously improve the self-flower fruiting capacity of apples and the fruit yield of apples by reducing the expression of the apple PCHR protein. When the method is applied to actual production, on one hand, the expression of PCHR protein can be inhibited in the flowering phase of apples so as to realize the effect of closing flower pollination, namely, fruit setting and high yield; on the other hand, the apple PCHR gene can also be mutated to obtain an apple strain with strong flower formation capability. The apple PCHR protein and the coding gene thereof and the corresponding inhibitor have great application value in improving the self-pollination fruiting capacity of self-parthenocarpic apples and the self-parthenocarpic molecular breeding of apples.
Drawings
The invention has the following drawings:
FIG. 1 shows the expression level of the PCHR gene in the columns 48h (a) and 72h (b) after pollination of the overexpressed PCHR gene and the expression level of the PCHR gene in the columns 48h (c) and 72h (d) after pollination of the silenced PCHR gene; wherein Mock represents the "Hanfu" (a, b) and "Fuji" (c, d) columns after self-pollination, respectively, without treatment, pCambia-1300 EV is the empty column injected with the over-expression vector, TRV EV is the empty column injected with the TRV of the VIGS system, OE PCHR is the over-expressed column injected with the PCHR gene of the VIGS system, and TRV PCHR is the silenced column injected with the PCHR gene of the VIGS system.
FIG. 2 is an observation of pollen tube growth after self-pollination of the "Hanfu" overexpressing PCHR gene (a) and "Fuji" silencing of the PCHR gene's columella (b); where Mock is the fluorescent observation of untreated flower columns for self-pollination, 48HAP and 72HAP represent 48 hours and 72 hours, respectively, after pollination.
FIG. 3 shows the results of fruit set phenotype and fruit set rate statistics 15 days after self-pollination of the over-expressed PCHR gene (a, b) and the silenced PCHR gene (c, d), N being the total number of flowers actually investigated.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
The PCHR gene of the present invention is preferably derived from apple 'Hanfu' (Malus domestica) and the gene coding sequence comprises 1263 nucleotides. The invention is described inPCHRThe expression level of the gene in plants is reduced, the calcium ion steady state in pollen tubes is enabled, and the growth of the pollen tubes is inhibited. Meanwhile, the PCHR gene also significantly affects the self-pollination seed setting rate of apples.
The application of malus self-flower fructification will be described in detail below. The following examples and figures are for illustrative purposes only and are not to be construed as limiting the scope of the present invention. Unless otherwise specified, reagents and consumables used in the examples described below were purchased commercially.
Example 1
Cloning of apple PCHR Gene
0.5g of pollinated flower column is taken, total RNA is extracted by using a plant polyphenol polysaccharide RNA extraction kit of Aidelai company, and cDNA is synthesized by using a cDNA synthesis kit of Aidelai company. The invention also provides a pair of primers for amplifying the PCHR gene, which are divided into forward primers as shown in SEQ ID NO. 3: 5'-ATGGCGTTTAGGGTTCGAAACAGC-3', the reverse primer is shown in SEQ ID NO. 4: 5'-TTAACACTTATGTTTCTCAGACTCCTCGG-3'.
The PCR method for amplifying the PCHR gene comprises the following steps: the apple cDNA is used as a template, the primer pair is used as a primer for PCR amplification, and a reaction system for PCR amplification comprises 10 mu L: 1. Mu.L of template DNA, 5. Mu.L of Taq mix, 0.5. Mu.L of forward primer, 0.5. Mu.L of reverse primer, ddH 2 O3. Mu.L; the concentration of cDNA is regulated to 100 ng/. Mu.L, and the concentrations of the forward primer and the reverse primer are 10 mu.M; the PCR amplification procedure includes: 95 ℃ for 5min;95 ℃ 30s,60 ℃ 30s,72 ℃ 90s,35 cycles; 72 ℃ for 7min and 12 ℃. Detecting PCR amplified products by agarose gel electrophoresis, recovering and purifying target fragments by using a Thermo Fisher Scientifi gel recovery kit, connecting the recovered fragments to a Takara pMD19-T cloning vector, transforming DH5 alpha escherichia coli competence, culturing on an ampicillin resistant medium, picking monoclonal, and detecting colony PCR to obtain the monoclonal strain with the target fragments.
Example 2
PCHRConstruction of Gene overexpression and silencing vectors
The overexpression vector used in this example was pCambia1300-eGFP, and the gene silencing was performed by the VIGS technique, and the vector was tobacco embrittlement Virus (Tobacco rattle virus, TRV) TRV viral vector. The CDS full length and +456 to +891 fragments were used as over-expression and VIGS vector construction sequences, respectively.
The following primers were designed to amplify the full-length CDS fragment for over-expression vector construction:
1300-PCHR-F：5’-CGATACACCAAATCGACTCTAGAATGGCGTTTAGGGTTCGA-3’ (SEQ ID NO .5)
1300-PCHR-R：5’-GCCCTTGCTCACCATGGTACCACACTTATGTTTCTCAGACT-3’ (SEQ ID NO .6)。
the following primers were designed to amplify gene silencing homologous sequence fragments for TRV vector construction:
TRV2-PCHR-F：5’-GTTACCGAATTCTCTAGAAGCACCAAGAAAGTTCACGC-3’ (SEQ ID NO .7)。
TRV2-PCHR-R：5’-ACGCGTGAGCTCGGTACCGGGTTGTCAATCAGTGGTGC-3’ (SEQ ID NO .8)。
the amplified fragment was ligated into the TRV2 vector after double digestion with XbaI/KpnI by homologous recombination. E.coli transformed with DH5 alpha, the obtained product is spread on LB solid medium with 50mg/l, cultured for 1 day at 37 ℃ and then picked up for PCR detection, and the monoclonal bacterial strain with target plasmid is obtained.
Example 3
PCHRFunction of gene in regulating and controlling self-flower fructification of apples
The pCambia1300-eGFP-PCHR and pTRV2-PCHR plasmids carrying the fragments of interest were transformed into GV3101 Agrobacterium competence, which can be purchased commercially and are described in detail in the specification. After the transformation is finished, the bacterial liquid is coated on a YEB solid culture medium containing 50mg/L rifampicin and 50mg/L kanamycin, the bacterial liquid is cultured for 2 days at 28 ℃, a plurality of monoclonal antibodies are selected for colony PCR detection, single colonies which are successfully transformed are selected and added into the YEB liquid culture medium containing 50mg/L rifampicin and 50mg/L kanamycin, the bacterial liquid is cultured for 2 days under the condition of 28 ℃ in an oscillating way, and the bacterial liquid is used for subsequent injection experiments after turbidity.
And centrifuging the bacterial liquid at 6000 rpm, re-suspending bacterial cells by using the contained heavy suspension, and standing for 4 hours to carry out injection experiments. The experiments were divided into 4 groups and the overexpression group was the strain with pCambia1300-eGFP-PCHR and the corresponding control with pCambia1300-eGFP empty strain; the resuspension bacteria with pTRV1 and pTRV2-PCHR were mixed in a ratio of 1:1 as a treatment group for the gene silencing experiment, and the resuspension bacteria with pTRV1 and pTRV2 empty were mixed in a ratio of 1:1 as a control group for the gene silencing experiment.
In the flowering period of apples, firstly, collecting self-bearing apples 'Hanfu' (HF) and self-bearing apples 'Fuji' (Fuji) pollen for self-pollination, collecting flower columns of two varieties after pollination for 48h and 72h, fixing the flower columns in FAA fixing solution, dyeing pollen tubes growing in the flower columns by adopting an aniline blue dyeing method, observing the growth condition of the self-bearing pollen tubes by an ultraviolet microscope, and finally counting the fruit setting rate.
And simultaneously, selecting flowers close to a big balloon period, injecting the prepared re-suspension bacteria liquid into the base part of a flower column, hanging a label beside an inflorescence after the injection, marking, and covering a black bag for light-shielding cultivation for 1 day. Picking off the fruit bag, carrying out artificial self-pollination on the post after injection, and sleeving a white fruit bag after pollination is finished. The flower columns after the partial treatment are collected after 48 hours and 72 hours of pollination are immediately put into liquid nitrogen, total RNA is extracted in a laboratory by using a plant polyphenol polysaccharide RNA extraction kit of Aidelai company, cDNA is obtained through reverse transcription reaction, and the expression change of PCHR genes is detected by a real-time fluorescence quantification method. As shown in FIG. 1, after self-pollination of the flower column injected with no-load fungus liquidPCHRThe gene expression quantity is not obviously different from that in untreated flower columns, the higher PCHR gene expression quantity is detected by injecting the flower column with the over-expressed PCHR gene bacterial liquid in 48 hours and 72 hours after pollination, and the expression quantity is obviously higher than that of a control after pollination. The expression level of PCHR gene in the flower columns 48h and 72h after pollination of the VIGS silent PCHR gene is obviously lower than that of the control group, and the expression level is reduced to about 0.6 times of that of the no-load control.
And simultaneously, collecting the pollen posts of adjacent flowers on the corresponding inflorescences, fixing the pollen posts in FAA solution, dyeing pollen tubes growing in the pollen posts in a laboratory by adopting an aniline blue dyeing method, and observing the pollen tubes by an ultraviolet microscope. The results are shown in FIG. 2, and are shown to be over-expressed compared to the empty injection controlPCHRThe pollen tube growth in the pollen column of the gene is obviously inhibited, and only 48h and 72h after pollination can be performedVery few pollen tubes were observed that grew normally. No good growing flower columns are still observed after 72h of no-load self-pollination by injecting TRV into self-parthenocarpic 'Fuji' flower columns, and silencing is still observedPCHRThe pollen tube growing normally is observed in the 48h flower column after the gene self-pollination, and the pollen tube grows more than one third of the flower column after 72h and almost reaches the basal part.
And taking a picture after self-pollination for 15 days, recording the fruit setting state, and counting the fruit setting rate. The results are shown in fig. 3a, b: the self-fructification rate of the over-expressed PCHR gene in the flower columns is reduced from 52.17% and 42.11% to 19.48% respectively compared with an untreated group (Mock HF×HF) and an injection no-load group (pCambia 1300-EV) by taking the self-fructified variety 'Hanfu' (HF) as a test material. The self-anthesis fecundity variety 'Fuji' is used as a test material, and compared with an untreated group (Mock Fuji×Fuji) and an injection empty group (TRV EV), the PCHR gene is silenced in a flower column, and the self-anthesis fecundity rate is respectively improved from 4.49% and 3.68% to 27.83%.
What is not described in detail in this specification is prior art known to those skilled in the art.
Claims (2)
1. The application of the inhibitor of the coding gene of the PCHR protein of the malus plant in improving the self-flower setting capacity of the malus is characterized in that:
the amino acid sequence of the PCHR protein is shown as SEQ ID NO. 1;
the inhibition factor is gRNA shown in SEQ ID NO. 2;
the application is as follows: the transcription level of the coding gene of the PCHR protein of the malus is inhibited by the inhibiting factor of the coding gene of the PCHR protein of the malus with the amino acid sequence shown as SEQ ID NO.1 so as to improve the self-fruiting capacity of the malus.
2. A method for improving the self-flower setting capability of apples is characterized in that gRNA with a nucleotide sequence shown as SEQ ID NO.2 is adopted to inhibit the transcription level of a coding gene of PCHR protein of malus plants with an amino acid sequence shown as SEQ ID NO.1, so that the growth of pollen tubes for promoting the self-pollination of apples is realized, and the fertilization success rate and the fruit setting rate of the self-pollination are improved.
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| BRPI1008725A2 (en) * | 2009-02-25 | 2016-03-08 | Basf Plant Science Co Gmbh | methods for enhancing and improving plant yield characteristics relative to control plants, and for the production of a transgenic plant, construct, construct uses, a nucleic acid, and a nucleic acid sequence, plant, part of the plant or plant cell, transgenic plant, harvestable parts, products, isolated nucleic acid molecule, and isolated polypeptide |
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