CN117384903A - Yak Lnc4047 gene and application thereof - Google Patents
Yak Lnc4047 gene and application thereof Download PDFInfo
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Abstract
The invention provides a yak Lnc4047 gene and application thereof, and belongs to the technical field of genetic engineering. The nucleotide sequence of the yak Lnc4047 gene is shown as SEQ ID No. 1. The invention discovers the regulation and control effects of the Lnc4047 gene on the proliferation and myogenic differentiation of the yak myoblasts for the first time, namely that the Lnc4047 gene inhibits the proliferation of the myoblasts, but has a certain promotion effect on the myogenic differentiation. Provides an important basis for deep understanding of the muscle development mechanism of the cattle and provides a theoretical basis for rapid cultivation and meat quality improvement of new varieties of high-quality yaks.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a yak Lnc4047 gene and application thereof.
Background
Yak (Bos grunniens) is a herbivorous ruminant belonging to the class of mammalia, the family of cattle, the subfamily of cattle, and is living in high-altitude areas of high cold and low oxygen of three to six kilometers throughout the year. The yaks are used as mammals living on the plateau area, and the distribution area has the characteristics of low air temperature (annual average is less than or equal to 0 ℃), large day-night temperature difference (more than 15 ℃), short grass growing season (110-135 d), strong radiation (annual radiation quantity is more than 140-195 KJ/cm < 2 >), low oxygen partial pressure (less than 110mm Hg) and the like, and the natural environment is very harsh. Due to the special ecological environment and the extremely strong natural selection, the yaks have a stubborn vitality and a set of unique physique morphological structure and physiological mechanism for coarse material resistance and cold resistance. This makes yaks not only greatly different from other bovine species in terms of body performance and physique, but also different in terms of meat yield, meat quality, and nutritional ingredients contained in meat products. In 2019, the yaks were slaughtered nationally about 360 ten thousand, the carcass weight average was about 128 kg/head, the carcass yield was about 47 ten thousand tons, the net meat yield was 37 ten thousand tons, and the yak meat yield value was estimated to be 270 gigabytes. In recent years, yaks become green foods popular in the public because of delicious meat quality, abundant nutrient substances, and rich various amino acids, high protein and low fat. But the special living environment and the withering period of 7 months lead the growth and weight increment of the plant to have wave-type growth, thereby influencing the economic value and the use value of the plant. The growth speed and meat production performance of livestock are closely related to the growth and development of skeletal muscle, and with the rapid development of the second generation sequencing technology, hundreds of thousands to millions of DNA molecules can be sequenced at one time by high-throughput, rapid and low-cost sequencing, so that genome or transcriptome sequencing becomes convenient and easy. Transcriptome sequencing is the basis of gene function and structure research, can solve the problems of deep excavation of new genes, low-abundance transcript discovery, transcription map drawing, gene family identification, evolution analysis and the like, and is widely applied to a plurality of fields such as biology, medicine, agriculture and the like. Therefore, the identification of the major genes affecting the growth traits of the yaks by using the novel technical means is particularly important for the healthy development of the yak industry.
The production characteristics of the yaks are mainly reflected in the growth and development of muscles, and the growth speed and the quantity of the muscles, especially the growth characteristics of skeletal muscles, directly influence the yield of the yaks. Skeletal muscle development is a complex physiological process, involves a series of developmental events, is the result of the combined action of environmental factors and genetic factors, involves a large number of transcription regulatory factors, signal pathways and complex regulatory mechanisms in the process, and has very important influence on the growth and development of animals. The proliferation and differentiation of myoblasts are research hot spots in recent years, skeletal muscles are not only components of animal growth and development, but also bear animal energy metabolism vectors, and research finds that lncRNAs are involved in skeletal muscle development and fiber type conversion, and the research is mainly focused on human and mice, but reports about research on functions and mechanisms of 1ncRNAs related to livestock skeletal muscle development are relatively few, the number of 1ncRNAs is huge, the mechanisms of action are various, and regulatory mechanisms in different organisms are quite different, so that it is important to accurately determine the action mechanisms of 1ncRNAs in different species.
Disclosure of Invention
In order to solve the problems, the invention provides a yak Lnc4047 gene and application thereof, and the invention discovers that the Lnc4047 gene has a regulation effect on proliferation and myogenic differentiation of myoblasts of yaks for the first time, namely, the Lnc4047 gene inhibits the proliferation of myoblasts, but has a certain promotion effect on myogenic differentiation. Provides an important basis for deep understanding of the muscle development mechanism of the cattle and provides a theoretical basis for rapid cultivation and meat quality improvement of new varieties of high-quality yaks.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a yak Lnc4047 gene, and the nucleotide sequence of the yak Lnc4047 gene is shown as SEQ ID No. 1.
The invention also provides application of the yak Lnc4047 gene in regulation and control of proliferation of yak myoblasts.
Preferably, the overexpression of the yak Lnc4047 gene inhibits proliferation of yak myoblasts.
Preferably, silencing the yak Lnc4047 gene promotes proliferation of yak myoblasts.
The invention also provides application of the yak Lnc4047 gene in regulating and controlling the differentiation of the yak myoblasts.
Preferably, the overexpression of the yak Lnc4047 gene promotes differentiation of yak myoblasts.
Preferably, the silenced yak Lnc4047 gene inhibits yak myoblast differentiation.
The beneficial effects are that:
the invention discovers the regulation and control effects of the Lnc4047 gene on the proliferation and myogenic differentiation of the yak myoblasts for the first time, namely that the Lnc4047 gene inhibits the proliferation of the myoblasts, but has a certain promotion effect on the myogenic differentiation. Provides an important basis for deep understanding of the muscle development mechanism of the cattle and provides a theoretical basis for rapid cultivation and meat quality improvement of new varieties of high-quality yaks.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows purification of primary skeletal myoblasts of yaks by the Percoll method;
FIG. 2 shows the expression of Lnc4047 gene in cell proliferation and differentiation stages and the nuclear separation expression profile at different stages;
FIG. 3 shows the effect of CCK8, edu and flow cytometry on proliferation of yak myoblasts by Lnc4047 gene;
FIG. 4 shows the changes in myodifferentiation marker gene MyF, myoG expression and protein abundance after interfering/overexpressing the Lnc4047 gene;
FIG. 5 shows the in situ levels of MyoG and MyF after immunofluorescence detection of interference/overexpression of the Lnc4047 gene.
Detailed Description
The invention provides a yak Lnc4047 gene, wherein the nucleotide sequence of the yak Lnc4047 gene is shown as SEQ ID No.1, and the method specifically comprises the following steps:
GACGGCTGTCACTCAGCCTGACGGACAGCCCAGAGATCGGCCTTCAGATAAAACCTTACAGATGTGGAGGTCGGTATTGATTTTGAGTTAGTAACGGTAAGGTACCAAGAAGTAATACATTAGTGAAAGCAAGGTCACTGGGAAAAAGGTAAAACCCACTGGGGGAGAGGCCAGGGCATTCTTGAGGGGGGTGATGACCGCATCAGATCAGGACTTCCTCAAGTTCAAAAGTTTGCAGGCCCCAGACCTCTGGTGGGCCCCTAACTGTGGGCCAACGCTTCCTGCTTCCCTGGGCGAAGGGGGACCCGTGGGGGTCTGTCAGCCGCCCGAGACTGGCGAGGGCCGTGTAGGCCTGTGGAGCTCTGCCCTGCGGGGCTGCCGTGGGCCTCTCTCCCCACGCTCTCTGGCTCAGCTTGCTGTGGGGAGGGATCACAGCGAGGGGtgaggagacacgggttcaagtcccagtctgccACGACCTGGCGGTGCAGTCTCAGGCACTCCTTTTCACGTCCCTGTGCCTtgatttccccatctgtagaatgggggtaAAACAGCTCATTTGCCGGAGAAGTCATTCCACGCTCCACCTCTCTTTAGAGGGGTCTGACCCCATCAGTTTGGAAGCGCTCATGCATGCCTTGAGACAGGAGGCTGGACGCAGGCAAAGGCCTCTGATCCCCAGACTTCCGGGCACCCTGGAAGAGAGCGAGCAGATGCTGCACACCCCCCATTCACACCCAGCCCAGATGTGGACCTGATTTGATGTTGCAGGGCAAATCCCACTGAACAAATCCCAGTACAGGGAAAGCTCCCCGGGCCTGAGCAGATTCTGAGCTGTCTGTTAATGGTGCAGGGTCCAAGCCCCCCCAAGCCACTGGGCACCACACGAACCCCTTCTCACAGAGCATTCCCGGAAGGTTAGCATcgaccttcttgctgtgtctcaGATCTGCTAGGCGTGTTCcagcctttgcacttgctggcTCTTCTGCCTGGACAACTCTCTGTTCTGTCTTCCCCTGGCTGATTCTTTCTGGTCT TGCTAGTGAGGTCTTCCCTGACTACTCcacatattttttcttccatagCACTTATCAGTATTGGAAATTATATTATCTGTTTTCATCTTCCTACAGTTGACTGACTGGCAGCTCCTGAAGGTCATGGAGCTGTTAGTCTTCATTCCTGAATTTCAATGCCTggtacagtgtctggcacatattaggtgctcaataaatatttgttaagtgaacATGCATGTATTTGACAGGGTTTTCTGAGCTTATAGTTTGCAGGGGGTACAGTGTAGCCACCGGGGCTGCAGTGATGGATGAAACACAATTTCATTCTGCCCTCAGAGAGCTCACAGCTGAGCCCTGTCTCCCGCCTGGCATATAATAACGCTCTGTGAAGTGTCTTATGAATGAAGGAAGGATGTCTTTAGGAAACACTATGTGGTTTGGGACAGCTGGGACAGAAAGTGCAAGgtaggaaggaggcagggatTTGGGACAGGCAAGTGGAGGTCCAGTCCCACGAAGCTTCGCCCTGTTCATCTGGGAGGTTCCAAAGAGCCACGGGTAATTCTTGAGCAACGTGGTCAGATTTGTTGGGCGGCTGTTGCCTTGCAGGATGAGTATGGATCTATGCACACCTTCCAGCCTGTCCTCCAAAGcatccttctcctctccccaaaTACCTCCCCACTTGTCTCCTCCATCCCATCCTTGCTGGGCCTGGCGTCTTGGTGATATGTAAAGAGAAGGATTATACCAGAGGCTCATCAGAACTGACAAACCCTCATGAGAGGCTTCCCACCCCACAACGATCctttcaaagaaagagaaatgggacCTGTGTGTTTTCCCTGAGCCTCTAAGTTTGTTCTTGGGGCCAGGGCTCCTTGAAGGAGGAGCAGTGTTAGCAATGAATCAGGTCTCCTAACCCCAGGGCAGTGTCAGCCTCCTGAGTGCCCCCGGCCCCGCAGacccaccaaacacacacacccagcaaACTCCTCAGGCCCAGGACCCCACCGCAACAGTCAGGAAAGAACAAACCCAGCTCCACCTGTGTTTCTGCCATCTCATTGGGCTGGCACCCTGCAAAGTCCCACCCAAACCTCACCAGCTGCGGTGGAGACAAAGTCCAGCCCCAGTAGGCCTGGTGCCCTTGACTTcagctctgggggtggggtgaggaaagGACAGAGGGGTTGAGAGAAAGGCTGAGAGGTGCACGCTGGGTGCATTGCAGGAAGAGTCAGGGGAGGCTGTGCTCGTGGTATTCTTGAGGAGCCAGGCCCCCTGCGGGCCTGGGatcccaccccacacccccaggGAACCCCCATCTGGAGTGAGAACCTCTATCATGGTCACGTGTCTAAAACCCATCCTTGAGAGTCAGACTTGACATCATCAGGCTAGAGGGGTCAAGCCCTGGCACACCCGCTGTGAGATCTGGGGAAGACGCACCTCTGGGTGGGCCTCGGTCtcttcatctgagaaatgggcGTCATGACTCCGCCCCACACGTCTCTGGAGGGCTCTGCACTGGCAGAGTCACGCCGAGTCTGTTGCTAAGAggagggaaagggaaagagg。
the invention also provides application of the yak Lnc4047 gene in regulation and control of proliferation of yak myoblasts. In the present invention, the overexpression of the yak Lnc4047 gene preferably inhibits proliferation of yak myoblasts. In the present invention, silencing the yak Lnc4047 gene preferably promotes proliferation of yak myoblasts.
The invention also provides application of the yak Lnc4047 gene in regulating and controlling the differentiation of the yak myoblasts. In the present invention, the overexpression of the yak Lnc4047 gene preferably promotes differentiation of yak myoblasts. In the present invention, the silencing of the yak Lnc4047 gene preferably inhibits yak myoblast differentiation.
The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.
Example 1
1. The separation method comprises the following steps:
neonatal calves were sacrificed according to standard procedures. The sampled hair and skin were removed to expose skeletal muscle tissue and tissue samples were transferred to 1 XPBS with 10% diabody using sterile surgical instruments. Rapidly transferring the sample into a sterile cell room, and removing connective tissues and blood vessels in the muscle by using a sterile surgical instrument. Samples were placed in an appropriate amount of 1 XPBS and the tissue was minced with sterile surgical instruments. An appropriate amount of collagenase type 4 solution is added to the minced tissue sample and the tissue is further minced in the presence of digestive enzymes. After the two shearing processes, the muscle tissue has become substantially chyme. The skeletal muscle minced tissue was then transferred to a centrifuge tube and digested for 20min in a 37 ℃ water bath. During the period, the tissue is digested more fully by lightly blowing with a pipette for multiple times.
2. The purification mode is as follows:
since the isolated skeletal muscle cells mainly include myoblasts and fibroblasts, a five-step Percoll gradient (fig. 1 a) was used in the invention. Collected cells with a Percoll fraction of 27.5-35%, 35-40% and 40-55% were defined as F1, F2 and F3, respectively. The expression levels of two myoblast marker genes (MyoD and c-met) and two fibroblast marker genes (FGF 7 and coll) were analyzed using qPCR. The results showed that the F3 fraction had higher levels of myoblast markers and lower levels of fibroblast markers (B-E in FIG. 1) compared to Fl and F2, suggesting the presence of purified myoblasts in F3. In addition, the differentiation status of these cellular components was examined. F3 is most abundant than the other components myotubes (F-H in fig. 1), suggesting the presence of myogenic cells in F3. Thus, it was demonstrated that Percoll density gradient centrifugation can purify myoblasts isolated from the longest muscle of the back of a yak without the need for any expensive equipment or special techniques.
3. Cell passaging and cryopreservation
Termination medium dmem+10% fbs (fetal bovine serum)
DMEM(sigma D6429) 45mL
FBS 5mL
Totally 50mL
Myoblast medium DMEM/F-12+20% FBS+1% double antibody (Green streptomycin)
The ratio of the frozen stock solution to the frozen stock solution is 3:1:1
Cell passage (1 pass 3)
The cells were observed under a microscope to confluence to about 80%, transferred to a super clean bench, medium in the dish was discarded, washed 2 times with D-PBS, 1 mL trypsin (bottom was required) was added, and the mixture was washed at 37℃with 5% CO 2 Digesting about 3 mm in an incubator, observing cells under a microscope to form a circle, stopping digestion, adding 2 mL termination culture medium, blowing the bottom, transferring cell sap into a 15 mL centrifuge tube, centrifuging at 1300r/min for 5 min, centrifuging, discarding supernatant, adding myoblast culture medium into the precipitate according to passage ratio, blowing, mixing uniformly, and uniformly distributing to a new culture dish or culture flask, cooling at 37deg.C in 5% CO 2 Culturing in an incubator, and changing the liquid once for two days.
Cell cryopreservation (1 pass 3)
The cells were observed under a microscope to confluence to about 80%, transferred to a super clean bench, medium in the dish was discarded, washed 2 times with D-PBS, 1 mL trypsin (bottom was required) was added, and the mixture was washed at 37℃with 5% CO 2 Digesting about 3 mm in an incubator, observing cells under a microscope to form a circle, stopping digestion, adding 2 mL termination culture medium, blowing the bottom, transferring cell sap into a 15 mL centrifuge tube, centrifuging for 5 min at 1300r/min, centrifuging, discarding supernatant, multiplying 500 mu L of DMEM/F-12 culture medium by passage ratio in sediment, blowing and mixing uniformly, adding 500 mu L of suspension into each freezing tube, adding 500 mu L of freezing solution, sealing with a sealing film, placing in a freezing box, placing in a refrigerator at-80 ℃ for 24h, and placing in a liquid nitrogen tank for long-term storage.
Cell distribution of Lnc4047 Gene
To examine the expression characteristics of the Lnc4047 gene at the growth and differentiation stages in myoblasts. These results suggest that Lnc4047 gene may be involved in proliferation and early differentiation of yak myoblasts as detected by qRT-PCR (FIG. 2A-B)
To analyze the nuclear cytoplasmic distribution of Lnc4047 gene, myoblasts in proliferation phase (Myoblast) and Myotube cells in differentiation phase (Myotube) were collected, respectively. After isolation of cytoplasmic RNA, mRNA expression levels of Lnc4047 gene in Myobast and Myotube were detected, respectively, wherein NEAT1 and GAPDH were used as control genes for specific expression of the nucleus and cytoplasm, respectively. The results of c qRT-PCR in FIG. 2 show that the Lnc4047 gene is distributed in both the nucleus and cytoplasm during the cell proliferation phase. However, as myogenic differentiation proceeds, the Lnc4047 gene content in the cytoplasm gradually increases from about 57% in Myoblast cytoplasm to 78% in Myotube cells (C-D in fig. 2). The subcellular localization of the Lnc4047 gene was examined using FISH technique, consistent with the results of Real-Time PCR, with Lnc4047 gene distributed in the undifferentiated cytoplasm, whereas in the differentiated cells, lnc4047 gene was mainly distributed in the cytoplasm (E in fig. 2).
Lnc4047 Gene inhibits proliferation of yak myoblasts
The effect of Lnc4047 gene on myoblast proliferation was examined using CCK-8, and the examination result showed that overexpression of Lnc4047 gene could inhibit myoblast proliferation, whereas silencing Lnc407 could increase myoblast proliferation capacity, compared to control group (a in fig. 3). Edu cell proliferation assay shows that when the Lnc4047 gene is overexpressed, the proliferation of cells is significantly reduced compared to the control, and when the Lnc4047 gene is silenced, the proliferation of cells is significantly increased, indicating that the Lnc4047 gene can inhibit cell proliferation (B in fig. 3).
Cell cycle is an important indicator for the characterization of cell proliferation. Different phases of cell development (G0, G1, S, G) can be distinguished by flow cytometry after staining with the fluorescent dye Propidium Iodide (PI) due to differences in DNA content. Cell cycle changes after overexpression/silencing of the Lnc4047 gene were detected using flow cytometry. The results showed that overexpression of the Lnc4047 gene significantly reduced the number of cells in S phase compared to the control, while silencing the Lnc4047 gene significantly increased the number of cells in S phase (C-H in fig. 3).
Lnc4047 Gene promotes differentiation of yak myoblasts
Myoblasts were infected with either the Lnc4047 gene over-expressed lentivirus or silenced lentivirus, and Real-time PCR was used to detect mRNA expression levels of Lnc4047 gene, myoG and MyF5 after 48h of infection. The results showed that the relative expression levels of Lnc4047 genes of group C and group E were significantly up/down-regulated after infection compared to the control group, indicating good infection efficiency. mRNA expression levels of differentiation marker genes MyoG and MyF5 in myoblasts were significantly increased after overexpression of the Lnc4047 gene compared to the control group, whereas mRNA expression levels of MyoG and MyF5 in myoblasts were significantly decreased after silencing of the Lnc4047 gene. After 48h of infection by Westernblot, the expression levels of Lnc4047 gene, myoG and MyF5 protein were examined. The results showed that the protein expression levels of MyoG and MyF5 in myoblasts were significantly increased after overexpression of the Lnc4047 gene compared to the control group, whereas the protein expression levels of MyoG and MyF5 in myoblasts were significantly decreased by silencing the Lnc4047 gene (fig. 4).
The results of detecting changes in MyoG and MyF5 protein levels after overexpression/silencing of the Lnc4047 gene using immunofluorescence showed that the levels of MyoG and MyF5 protein expression in myoblasts were significantly increased after overexpression of the Lnc4047 gene compared to the control group, whereas the levels of MyoG and MyF5 protein expression in myoblasts were significantly decreased after silencing of the Lnc4047 gene, indicating that the Lnc4047 gene was able to promote myoblast differentiation (fig. 5).
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 (7)
1. The yak Lnc4047 gene is characterized in that the nucleotide sequence of the yak Lnc4047 gene is shown as SEQ ID No. 1.
2. The use of the yak Lnc4047 gene of claim 1 in regulating proliferation of yak myoblasts.
3. The use according to claim 2 wherein the overexpression of the yak Lnc4047 gene inhibits proliferation of yak myoblasts.
4. The use according to claim 2 wherein silencing the yak Lnc4047 gene promotes yak myoblast proliferation.
5. The use of the yak Lnc4047 gene of claim 1 in regulating differentiation of yak myoblasts.
6. The use of claim 5 wherein the overexpression of the yak Lnc4047 gene promotes yak myoblast differentiation.
7. The use of claim 5 wherein silencing the yak Lnc4047 gene inhibits yak myoblast differentiation.
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Citations (3)
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| CN108753779A (en) * | 2018-06-07 | 2018-11-06 | 天津农学院 | Ox lncRNA-133a and the application in the regulation and control of bovine muscle satellite cell Proliferation, Differentiation and verification method |
| CN115976220A (en) * | 2023-02-27 | 2023-04-18 | 重庆医科大学国际体外诊断研究院 | Use of novel Lnc-FLJ for treating castration-resistant prostate cancer |
| WO2023102377A2 (en) * | 2021-11-30 | 2023-06-08 | Dana-Farber Cancer Institute, Inc. | Antisense inhibitors of mir17hg pre-rna as therapeutic agents in cancer |
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| CN108753779A (en) * | 2018-06-07 | 2018-11-06 | 天津农学院 | Ox lncRNA-133a and the application in the regulation and control of bovine muscle satellite cell Proliferation, Differentiation and verification method |
| WO2023102377A2 (en) * | 2021-11-30 | 2023-06-08 | Dana-Farber Cancer Institute, Inc. | Antisense inhibitors of mir17hg pre-rna as therapeutic agents in cancer |
| CN115976220A (en) * | 2023-02-27 | 2023-04-18 | 重庆医科大学国际体外诊断研究院 | Use of novel Lnc-FLJ for treating castration-resistant prostate cancer |
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