CN115896166B - Application of tea CsGS gene in regulating plant glutamine metabolism and promoting plant leaf growth - Google Patents
Application of tea CsGS gene in regulating plant glutamine metabolism and promoting plant leaf growth Download PDFInfo
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Abstract
The invention discloses an application of a tea tree CsGS gene in regulating plant glutamine metabolism and promoting plant leaf growth, wherein the ORF sequence of the CsGS gene is shown in SEQ ID NO. 1; the gene interfering CsGS gene expression is a precursor sequence of tea tree CsmiR396d gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2. The expression of the tea leaf CsGS gene is obviously up-regulated in CsGS2 over-expression and CsmiR396d interference, and the glutamine content is obviously increased; the CsGS gene expression in the tea leaves with CsGS d interference and CsmiR d over expression is obviously reduced, the glutamine content is obviously reduced, and the glutamine content can be regulated by regulating the CsmiR d and CsGS2 content in plants, so that the growth of the tea leaves is promoted.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to application of a tea CsGS gene in regulating plant glutamine metabolism and promoting plant leaf growth.
Background
Nitrogen (N) is one of the plant nutrients and plant availability of nitrogen is one of the important limiting factors for plant yield. The tea tree utilization part takes young bud leaves as the main part, when nitrogen is insufficient, the chlorophyll content in the young shoots is reduced, photosynthesis is weakened, and the tea yield is reduced; when nitrogen is sufficient, plants grow vigorously, the cycle of picking young shoots with rapid bud germination is increased, and the tea yield is increased. In addition, the nitrogen can increase the content of free amino acids, chlorophyll, water extract and aroma components in the tea, reduce the content of phenolic compounds and improve the quality of the tea.
In plants, the assimilation of inorganic nitrogen into organic nitrogen available to plants is mainly accomplished by Glutamine Synthetase (GS), which is the rate-limiting enzyme in nitrogen assimilation. In plants, GS can be divided into GS1 located in the cytosol and GS2 located in the plastids. Wherein, GS1 acts mainly on primary NH 4+ assimilation in roots and GS2 acts mainly on re-assimilation of NH 4+ in chloroplast photoplethysmography;
At present, miR396 is one of miRNA families conserved in plants, and has been found to play an important role in leaf development and adversity stress in other plants, but the function of miR396d in tea trees is not clear.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an application of a tea CsGS gene in regulating plant glutamine metabolism and promoting plant leaf growth.
In order to achieve the purpose, the invention designs application of a tea tree CsGS gene in regulating plant glutamine metabolism and promoting plant leaf growth, and an ORF sequence of the CsGS gene is shown as SEQ ID NO. 1.
Further, the plant is arabidopsis thaliana or camellia sinensis.
Still further, the tea CsGS gene was ligated to a vector, transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation, screened, cultured and a transgenic line was obtained.
The invention also provides an application of the interference CsGS gene expression in regulating the plant glutamine metabolism and promoting the plant leaf growth, wherein the gene interfering CsGS gene expression is a precursor sequence of the tea tree CsmiR396d gene, and the nucleotide sequence of the precursor sequence is shown as SEQ ID NO. 2.
Further, the plant is arabidopsis thaliana or camellia sinensis.
Still further, the precursor gene of the tea CsmiR396d gene was ligated to a vector, transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation, screened, cultured and a transgenic line was obtained.
Still further, the mature sequence of the tea CsmiR396d gene is shown in SEQ ID NO. 3.
Sequence table 1
The invention has the beneficial effects that:
According to the invention, the target gene of tea CsmiR396d is CsGS d through degradation group data, and a fluorescence quantitative result shows that the expression quantity in 9 tissues (buds, first leaves, third leaves, tender stems, mature leaves, old leaves, lateral roots, flowers and fruits) of tea tree is in opposite trend, so that the method accords with the law of miRNA negative regulation target gene in plants. The 5' RLM-RACE experiment confirms that the cleavage site of CsmiR396d to CsGS2 is 534 th base; transient expression experiments of tobacco leaves also confirm the shearing action of CsmiR396d on CsGS. By stable overexpression of CsmiR396d and CsGS2 genes in arabidopsis it was found that: after CsmiR d of over-expression, the expression of the GS2 gene in the arabidopsis leaves is obviously reduced, and the content of glutamine is obviously reduced; after CsGS is over-expressed, the expression of GS2 gene in arabidopsis leaves is obviously increased, and the content of glutamine is obviously increased. Further, the gene CsmiR d and the gene CsGS d are respectively over-expressed and interfered in tea leaves, the expression of the gene CsGS of the tea leaves is obviously up-regulated in CsGS2 over-expression and CsmiR396d interference, and the content of glutamine is obviously increased; the expression of CsGS gene in tea leaves with CsGS d interference and CsmiR d over expression is obviously reduced, the content of glutamine is obviously reduced, and the glutamine content can be regulated in plants CsmiR d and CsGS 2; thereby promoting the growth of tea leaves.
Drawings
FIG. 1 shows the positive identification of Arabidopsis CsmiR396d and CsGS2 overexpressing lines;
FIG. 2 is a graph showing the glutamine content of an overexpression line of Arabidopsis CsmiR396d and CsGS2 and the expression level of the related genes;
FIG. 3 is a graph of the results of a 5' RLM-RACE experiment;
In the figure, the red arrow is CsGS of the degradation site of 2, and the number represents the proportion of CsGS2 cleaved at the CsmiR396d target site to the sequenced sample;
FIG. 4 is a plot of interactions of tobacco transient system verification CsGS2 and CsmiR396 d;
in the figure, A is a graph of GFP fluorescence signals under various combinations of intense light, ultraviolet light and in vivo imagers;
b is a graph of relative fluorescence intensity in different combinations;
FIG. 5 is a graph showing changes in related genes after CsmiR396d and CsGS d, respectively, have been silenced;
FIG. 6 is a graph showing the change in glutamine content after CsmiR d and CsGS d, respectively, have been silenced;
FIG. 7 is a graph showing the changes in the related genes after CsmiR396d and CsGS d, respectively, have been overexpressed;
FIG. 8 is a graph showing the change in glutamine content after CsmiR d and CsGS d, respectively, have been overexpressed.
Detailed Description
The present invention is described in further detail below in conjunction with specific embodiments for understanding by those skilled in the art.
Example 1 discovery of Gene function
1. Extraction of genomic DNA of tea tree: genomic DNA was extracted from tea leaves using a plant DNA kit (ideley).
2. Extracting RNA of tea trees: RNA was extracted from tea leaves using a plant RNA kit (Edley).
Amplification of csmir396d and CsGS:
(1) The full length of the ORFs containing CsmiR396d precursor fragments (SEQ ID NO. 2) and CsGS2, respectively, were amplified and a homologous recombination linker (Tsingke) was added to the 5' end of the designed primer, the primer sequences are detailed in the following table.
The amplified fragment was subjected to homologous recombination with vector pBinRed subjected to double digestion with XbaI and HindIII, to obtain recombinant plasmids CsmiR396d and CsGS2, respectively.
(2) The recombinant plasmid is transferred into an agrobacterium strain GV3101 by a chemical conversion method, and a recombinant agrobacterium strain containing the recombinant plasmid CsmiR396d and the recombinant plasmid CsGS d is obtained by screening.
(3) The inflorescences of healthy arabidopsis thaliana with good conditions are infected and grown for about one month by using recombinant agrobacterium strains containing recombinant plasmid CsmiR396d and recombinant plasmid CsGS2 respectively, and the infection is repeated for 4-5 times every four days. The mature seeds are collected and are dried in a baking oven at 28 ℃, and then the seeds with red fluorescence of T 0 generation are obtained through screening by an ultraviolet lamp.
4. The expression levels of CsmiR396d and CsGS2 in the overexpressed Arabidopsis lines were examined by RT-PCR:
Total RNA in leaves of transgenic arabidopsis and wild type arabidopsis is extracted by using a plant RNA extraction kit, and reverse transcription is performed by using a reverse transcription kit (Simgen), wherein a reverse transcription primer of CsGS2 is a random primer, a reverse transcription primer of CsmiR396d is a stem-loop primer which is specially designed according to a sequence, an internal reference gene of fluorescence quantification CsGS2 is GAPDH, and an internal reference gene of CsmiR396d is U6.
Analysis was performed on a AB StepOne Plus real-time PCR system using a 2 XSybr GREEN QPCR mixture (PC 6102, edley) as follows: denaturation at 94℃for 3min; 10s at 94℃and 34s at 60℃annealing (40 cycles); annealing at 95 ℃ for 15s and at 65 ℃ for 1min; the temperature reached 95℃again at a rate of 0.3℃per second, and then annealed at 95℃for 15s. The relative fold change in gene expression was performed by the 2 -ΔΔCt method. Each PCR reaction was independently repeated at least three times. Primer sequences are shown in the following table.
The results show that: the expression levels of CsmiR d and CsGS2 in transgenic arabidopsis thaliana containing CsmiR396d and CsGS2, respectively, were both significantly increased (fig. 1), indicating successful transfer and stable expression of the CsmiR396d and CsGS2 genes of tea tree into arabidopsis thaliana.
| Primer name | Primer sequence (5 '-3') |
| CsU6-F | CGGGGACATCCGATAAAATTG |
| CsU6-R | GGACCATTTCTCGATTTGTGC |
| CsGAPDH-F | TTGGCATCGTTGAGGGTCT |
| CsGAPDH-R | CAGTGGGAACACGGAAAGC |
| CsmiR396d-stem | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAGTTC |
| CsmiR396d-F | TAGCCTTCCACAGCTTTCTT |
| CsmiR396d-R | GTGCAGGGTCCGAGGT |
| CsGS2-F1 | AAATAAACGCTACAAGGCTGCTG |
| CsGS2-R1 | ACGCCCAAATGACTTATCCG |
5. Detection of glutamine content in CsmiR396d and CsGS2 overexpressing Arabidopsis lines using triple quadrupole liquid chromatography-mass spectrometry
Extraction of CsmiR396d and CsGS2 overexpressing glutamine in Arabidopsis leaves was performed on a C18 reverse phase chromatography column (Agilent 250 mm. Times.4.6 mm. Times.5 μm) using a triple quadrupole liquid chromatograph QUANTIS (Thermo Fisher, TSQ Altis/TSQ Quantis). The mobile phases A and B are pure water and acetonitrile respectively, the sample injection amount is 10 mu L, the flow rate is 0.30mL/min, the mass spectrometer works in a positive ion mode, and the detection wavelength is 210nm. The elution procedure was: 0 to 5min, phase b increases linearly from 1% to 10%; linear increase to 50% at 7min and 80% within 1min; maintaining 80% at 8-13.5 min; at 14min, phase B was reduced to 1% and held for 6min. The glutamine in the sample is characterized according to the retention time and the ionic characteristics of the standard substance, and the sample is relatively quantified according to the peak area and the sample preparation data.
The results showed that both the amount of GS2 and the glutamine content were significantly increased in CsGS2 over-expressed plants compared to wild type arabidopsis, whereas the amount of GS2 was significantly reduced in CsmiR396d over-expressed plants and the glutamine content was completely undetectable (fig. 2).
Example 2 demonstrates the shear of CsmiR396d on CsGS2
1. The shear of CsmiR396d on CsGS d was verified in tea tree
The cleavage of CsGS2 by CsmiR396d was verified using the 5' RLM-RACE technique using FirstChoice RLM-RACE kit (Invitrogen, thermo FISHER SCIENTIFIC). Under the action of T4 RNA ligase, connecting the designed 5' -end joint with total RNA (more than 1000 ng) of tea tree, and then reversely transcribing into cDNA by using a random primer; the resulting cDNA was analyzed on agarose gel by two rounds of PCR amplification using 5 'terminal adapter primers and 3' terminal gene specific primers. The target bands obtained were purified using DNA gel extraction kit (Code No. DR01, aidlab, beijing, china), cloned into pTOPO-TA vector (Code No. CV14, aidlab, beijing, china), transformed into DH 5. Alpha. Chemically competent cells (Code No. G6016, shanghai, china) and sequenced to determine whether mRNA was the cleavage site for miRNAs. The 5' end sequences and 3' end specific primer information for the genes used in the 5' RLM-RACE experiments are shown in the following table.
| Primer name | Primer sequence (5 '-3') |
| 5’RACE-adapter | GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA |
| RACE outer primer | GCTGATGGCGATGAATGAACACTG |
| RACE inner primer | CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG |
| CsGS2-out | TCATTTCCTTCTCCATAAGCACTG |
| CsGS2-in | CTCCGTTGGTGCCACTAATG |
After sequencing the resulting gene fragments, 2 target bands out of 20 sequencing results demonstrated that CsmiR396d can degrade part CsGS2 in the tea plant by complementation with the target site (FIG. 3).
2. Verification of CsmiR396d shear on CsGS d in tobacco
The MIR gene CsmiR d is inserted into the pBI121 vector after double digestion of XbaI and SacI by using a homologous recombination method. Two complementary target site sequences (21 nt) are synthesized respectively, and double-stranded DNA formed by annealing the two oligonucleotide DNA is inserted between two enzyme cutting sites XhoI and XbaI of the pMS4 vector by using a homologous recombination method. The recombinant plasmid is transferred into an agrobacterium strain GV3101 by a chemical conversion method, and a recombinant agrobacterium strain containing the recombinant plasmid CsmiR396d and the recombinant plasmid CsGS d is obtained by screening. Agrobacteria were combined two by two and infiltrated into the tobacco leaf from the back through a disposable needle. Under normal culture conditions, the expression of GFP fluorescence was observed 3 days later using a hand-held ultraviolet analyzer (ZF-5) and a living body imager (NIGHTSHADE L985, berthold). The primers used for construction of the MIR gene for CsmiR396d and the CsGS target site are shown in the following table.
| Primer name | Primer sequence (5 '-3') |
| CsmiR396d-HR-F | GAGAACACGGGGGACTCTAGACATAGGTGGGGAAAAGGTAGG |
| CsmiR396d-HR-R | GGGGAAATTCGGAGCTCCAAAAAAGCATCAAAATAATCCA |
| CsGS2-HR-F | TCCTCGGCCGAATTCCTCGAGCAGTAACAAGAAGGTTGTGGA |
| CsGS2-HR-R | TTCTTCTCCTTTACTTCTAGATCCACAACCTTCTTGTTACTG |
As a result, only CsmiR396d and CsGS2 bacteria were mixed, and CsmiR396d was degraded by binding to CsGS2 target site, thereby inhibiting GFP gene expression and reducing green fluorescent signal on tobacco leaves (FIG. 4).
Example 3CsmiR396d and CsGS2 Gene silencing
Synthesis of antisense oligonucleotides to csmir396d and CsGS (Tsingke): the sense and antisense oligonucleotide sequences of CsmiR396d and CsGS2 were designed using Soligo software (http:// sfold. Wadsworth. Org/cgi-bin/index. Pl.), the sequence information being given in the following table.
Transient silencing expression of csmir396d and CsGS2 on tea leaves: the solution containing the sense oligonucleotide and the antisense oligonucleotide is respectively injected to the left side and the right side of the leaf of the same tea tree, under normal culture conditions, the leaf of a control group on the left side and the leaf of a silencing group on the right side are respectively collected after 6 days, immediately frozen by liquid nitrogen after being picked, and stored in a refrigerator at the temperature of minus 80 ℃ for standby.
3. Detecting the expression quantity of CsmiR d and CsGS2 in tea leaves by RT-PCR, extracting total RNA in a tea leaf control group and a silencing group by using a plant RNA extraction kit, and performing reverse transcription by using a reverse transcription kit (Simgen), wherein a reverse transcription primer of CsGS2 is a random primer, a specific stem-loop (stem-loop) primer is designed according to a sequence by the reverse transcription primer of CsmiR396d, and the internal reference gene of fluorescent quantification CsGS2 is GAPDH and the internal reference gene of CsmiR396d is U6. The results showed that in CsmiR396d silenced leaves, csmiR396d expression was significantly reduced and CsGS2 expression was significantly increased (fig. 5A); in CsGS 2-silenced leaves, the expression level of CsGS2 was significantly reduced and the expression level of CsmiR396d was significantly increased (fig. 5B).
4. Detection of glutamine content in CsmiR396d and CsGS2 silenced tea leaves using triple quadrupole liquid chromatography-mass spectrometry
Extraction of glutamine from CsmiR396d and CsGS2 silenced tea leaves the sample was characterized for glutamine based on retention time and ionic characteristics of the standard using a triple quadrupole liquid chromatograph/mass spectrometer QUANTIS (Thermo Fisher) and relative quantification of the sample was based on peak area and sample preparation data. Results CsmiR396d silenced tea leaves showed a significant increase in glutamine content and CsGS2 silenced tea leaves showed a significant decrease in glutamine content (fig. 6).
Example 4CsmiR396d and CsGS2 Gene overexpression
1. Construction of the over-expression vector: the attB recombination linker (Tsingke) was added before the amplified CsMIR d and CsGS2 genes, and the primer sequences are shown in the following table.
The amplified fragments are inserted into an expression vector pMDC and 43 after BP and LR recombination reaction, and the recombinant plasmids of CsMIR396d and CsGS2 are obtained.
The pMDC43 vector plasmid and the recombinant plasmid were transferred into the Agrobacterium strain GV3101 by chemical transformation, and the recombinant Agrobacterium strains containing CsmiR396d and CsGS2, respectively, were obtained by selection.
Transient overexpression of csmir396d and CsGS2 on tea leaves: the solution containing pMDC empty agrobacterium strain and CsmiR396d recombinant agrobacterium strain, pMDC empty agrobacterium strain and CsGS recombinant agrobacterium strain is respectively injected to the left and right sides of the same tea tree leaf, under normal culture condition, 6 days later, leaf blades of a left control group and a right over-expression group are respectively collected, immediately frozen by liquid nitrogen after being picked, and stored in a refrigerator at-80 ℃ for standby.
3. Detection of the expression levels of CsmiR396d and CsGS2 in tea leaves by RT-PCR
And extracting total RNA in the tea leaf control group and the silencing group by using a plant RNA extraction kit, and performing reverse transcription by using a reverse transcription kit (Simgen), wherein a reverse transcription primer of CsGS2 is a random primer, a reverse transcription primer of CsmiR396d is a stem-loop (stem-loop) primer which is specially designed according to a sequence, an internal reference gene of fluorescence quantification CsGS2 is GAPDH, and an internal reference gene of CsmiR396d is U6.
The results show that: in the CsmiR396d overexpressed leaf, the CsmiR396d expression level was significantly up-regulated and CsGS2 expression level was significantly reduced (fig. 7A); in the CsGS2 overexpressed leaf, the expression level of CsGS2 was significantly increased and the expression level of CsmiR396d was significantly decreased (fig. 7B).
4. Detection of glutamine content in tea leaves overexpressed by CsmiR396d and CsGS2 using triple quadrupole liquid chromatography-mass spectrometry
Extraction of glutamine from tea leaves overexpressed by CsmiR396d and CsGS2 the sample was characterized for glutamine based on retention time and ionic characteristics of the standard and relative quantification based on peak area and sample preparation data using a triple quadrupole liquid chromatograph-mass spectrometer QUANTIS (Thermo Fisher).
The results are shown in the following: the glutamine content in the tea leaves overexpressed by CsmiR d was significantly reduced and the glutamine content in the tea leaves overexpressed by CsGS d was significantly increased (fig. 8).
When the recombinant agrobacterium strain CsGS2 transfects tea tree CsGS2 for over-expression, the expression quantity of CsGS2 is obviously increased, the expression quantity of CsmiR396d is obviously reduced, so that the glutamine content in tea tree leaves is obviously increased, and the growth of the tea tree leaves is promoted;
when the recombinant agrobacterium strain CsmiR d transfects tea trees, the expression quantity of CsmiR396d is obviously up-regulated, and the expression quantity of CsGS2 is obviously reduced, so that the glutamine content in tea tree leaves is obviously reduced, and the growth of the tea tree leaves is promoted; the balance of nitrogen nutrition of tea trees is affected, and the growth of plant leaves is reduced.
Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (3)
1. Use of a gene that interferes with CsGS gene expression for regulating glutamine metabolism in a plant, characterized in that: the gene interfering CsGS gene expression is a precursor sequence of tea tree CsmiR396d gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2; the plant is Arabidopsis thaliana or tea tree.
2. The use according to claim 1, characterized in that: the precursor gene of the tea CsmiR396d gene is connected to a carrier, and the transgenic strain is obtained through agrobacterium-mediated transformation into arabidopsis thaliana, screening, culturing and obtaining.
3. The use according to claim 1, characterized in that: the mature sequence of the tea CsmiR396d gene is shown in SEQ ID NO. 3.
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