阅读说明：本技术 尼克酰胺在改善泌乳期奶山羊乳成分中的应用 (Application of nicotinamide in improving milk components of lactating goats ) 是由 李君� 杨盛茹 许会芬 权凯 韩浩园 王笑笑 车龙 赵金艳 魏红芳 哈斯通拉嘎 于 2021-08-31 设计创作，主要内容包括：本发明涉及一种尼克酰胺在改善泌乳期奶山羊乳成分中的应用,分别从整体、细胞与分子水平出发,系统揭示尼克酰胺在调控奶山羊乳腺脂肪酸代谢中的作用机制。本发明发现尼克酰胺对奶山羊乳成分产生有益影响,将尼克酰胺应用于泌乳期奶山羊日常饲料中,能够提高奶山羊的产奶量,提高羊奶多不饱和脂肪酸的组成比例。本发明既提高了奶山羊的产奶量,又改善了羊乳品质,提高奶山羊养殖的经济效益。本发明阐明了尼克酰胺调控奶山羊乳腺脂肪酸代谢的作用机制,为尼克酰胺在提高奶山羊乳品质中的应用及深入研究奶山羊乳腺脂肪酸代谢调控网络提供理论和实验依据。(The invention relates to an application of nicotinamide in improving milk components of a dairy goat in a lactation period, and discloses an action mechanism of the nicotinamide in regulating and controlling the mammary gland fatty acid metabolism of the dairy goat systematically from the whole, cell and molecular levels. According to the invention, the beneficial influence of the nicotinamide on the milk components of the dairy goat is found, and the nicotinamide is applied to the daily feed of the dairy goat in the lactation period, so that the milk yield of the dairy goat can be increased, and the composition proportion of polyunsaturated fatty acid in the goat milk can be increased. The invention not only improves the milk yield of the milk goat, but also improves the quality of the goat milk and improves the economic benefit of breeding the milk goat. The invention clarifies the action mechanism of Nikeamide for regulating and controlling the fatty acid metabolism of the mammary gland of the dairy goat, and provides theoretical and experimental basis for the application of Nikeamide in improving the milk quality of the dairy goat and the deep research on a fatty acid metabolism regulation and control network of the mammary gland of the dairy goat.)

1. An application of nicotinamide in improving milk yield of milk goats in lactation period is provided.

2. Use of the nicotinamide according to claim 1 for improving the milk content of lactating goats.

3. The use as claimed in claim 2 wherein the milk component comprises milk fat percentage and milk fatty acid.

4. Use as claimed in claim 3 wherein the milk fatty acids include C4:0, C16:0 and C18: 1.

5. The use of the nicotinamide of claim 1 for regulating transcription of the transcription factor SREBP1 in FASN gene transcription.

6. The use according to any one of claims 1 to 5, wherein the amount of nicotinamide added to the milk goat basal diet is 5 g/d.

Technical Field

The invention relates to a new application of nicotinamide, in particular to an application of nicotinamide in improving milk components of a dairy goat in a lactation period.

Background

Nicotinamide (NAM) is a B-group vitamin with simple structure and stable physicochemical property, is an amide form of vitamin B3, and can form coenzyme I and coenzyme II with ribose, phosphate and adenine in animal body to participate in metabolism of lipid, carbohydrate and protein in vivo. NAM is naturally present in animal by-products and plant-derived feed materials. NAM is absorbed mainly through the mucosa of the gastrointestinal tract into the body for metabolism. Sirtuin-2 related enzyme 1(SIRT1) is NAD⁺Dependent deacetylases, which are key upstream regulatory factors regulating lipid metabolism, modify lysine residues of proteins through deacetylation to regulate physiological processes. The activity of SIRT1 can be inhibited by Nicotinamide (NAM), which acts mainly by inhibiting the activity of SIRT1 deacetylase, and the formation of fat is achieved not only by regulating the expression of transcription factors for the formation of fat, but also by regulating the expression of genes during the oxidation of fatty acids.

The goat milk has rich nutrition value, and especially has unique advantages in the aspect of milk fat. On one hand, the milk fat is smaller in droplet and is easier to digest and absorb; on the other hand, the goat milk has higher content of short-chain and medium-chain fatty acids. The milk fat is one of the main components of the goat milk, the fat content and the fatty acid composition of the milk fat are main reasons influencing the nutritive value and the flavor of the goat milk, the special flavor (namely the mutton smell) of the goat milk is a key restriction factor of the development of the goat milk industry and the expansion of the consumption market, and if the generation of the special flavor can be changed, the mouthfeel of the goat milk is improved, and the development of the goat milk industry is certainly promoted. Therefore, the deep research on the influence of nutrient substances on the fat content and the fatty acid composition of the goat milk and the milk fat metabolism regulation mechanism of the mammary gland of the goat have important theoretical and practical significance for further developing the goat milk consumption market and promoting the development of the goat milk industry.

The nicotinamide is used as premix of ruminant or as feed additive, the ruminant has different nutrition requirements in different physiological periods and different physiological states, and the nutrition regulation purpose of the nicotinamide is different. Therefore, the regulation mechanism, the effective regulation dosage, the addition mode, the action period and the like of the nicotinamide are fully understood, and the nutrient regulation means can be more reasonably utilized by people, so that the accurate nutrient regulation is expected to be realized. In order to further improve the milk quality of the milk goats and improve the flavor of the milk of the goats, the invention tries to add nicotinamide into the basic ration of the milk goats so as to influence the milk yield, milk components and other properties of the milk goats.

Disclosure of Invention

The invention aims to provide an application of nicotinamide in improving milk components of a dairy goat in a lactation period, and systematically discloses an action mechanism of the nicotinamide in regulating and controlling mammary gland fatty acid metabolism of the dairy goat from the whole, cell and molecular levels.

In order to achieve the purpose, the invention adopts the technical scheme that:

an application of nicotinamide in improving milk yield of milk goats in lactation period is provided.

The nicotinamide is applied to improving the milk components of the dairy goat in the lactation period.

The milk component includes a milk fat ratio and milk fatty acids.

The milk fatty acids include C4:0, C16:0, and C18: 1.

The use of said nicotinamide for the control of the transcription factor SREBP1 in the transcription of the FASN gene.

The addition amount of the nicotinamide in the basic ration of the milk goat is 5 g/d.

The invention has the beneficial effects that:

according to the invention, the beneficial influence of the nicotinamide on the milk components of the dairy goat is found, and the nicotinamide is applied to the daily feed of the dairy goat in the lactation period, so that the milk yield of the dairy goat can be increased, and the composition proportion of polyunsaturated fatty acid in the goat milk can be increased.

The invention not only improves the milk yield of the milk goat, but also improves the quality of the goat milk and improves the economic benefit of breeding the milk goat.

The invention clarifies the action mechanism of Nikeamide for regulating and controlling the fatty acid metabolism of the mammary gland of the dairy goat, and provides theoretical and experimental basis for the application of Nikeamide in improving the milk quality of the dairy goat and the deep research on a fatty acid metabolism regulation and control network of the mammary gland of the dairy goat.

Drawings

FIG. 1 is a graph showing the effect of different concentrations of NAM on mRNA expression of important signal molecules in the SIRT1/FOXO1 pathway.

FIG. 2 is a graph showing the effect of 300. mu. mol/L NAM on milk fat synthesis-related gene expression.

FIG. 3 is a graph showing the effect of NAM on triglyceride levels and lipid droplet aggregation in mammary epithelial cells of a dairy goat.

Figure 4 is a graph of the effect of NAM on fatty acid composition in mammary epithelial cells.

FIG. 5 is a schematic representation of potential transcription factor binding sites of the FASN gene promoter.

FIG. 6 is a graph of the effect of SIRT1 agonists and inhibitors on FASN promoter activity.

FIG. 7 is a schematic diagram showing the sequence alignment of the SRE1 and SRE2 mutations.

FIG. 8 shows the effect of SRE1 and SRE2 mutations on FASN promoter activity.

FIG. 9 shows the effect of NAM on the activity of FASN wild-type and SRE mutant promoters.

Detailed Description

The following examples further illustrate the embodiments of the present invention in detail.

The embodiment of the invention comprises the following data processing: the experimental data are expressed as mean ± standard deviation, and the significance of the difference is analyzed by t-test comparing the mean of two samples by SPSS software. P <0.05 indicates significant difference, and P <0.01 indicates very significant difference.

Example 1 Experimental design and feeding management of milk goats

40 dairy goats with similar gestation times (2 gestation times), similar lactation days (30 +/-5 days), same or similar weights and good health conditions are selected and randomly divided into two groups (a control group and a test group), wherein each group comprises 20 goats, the control group is fed with basic diet, and the test group is added with 5g/d of Nicotinamide (NAM) on the basis of the basic diet. The experimental diet design was referenced to NRC (2007) goat breeding standards, and the basal diet composition and nutritional ingredients are shown in tables 1 and 2. The whole test period is 60 days, wherein the pre-test period is 10 days, and the positive test period is 50 days. The test was carried out in a certain milk goat farm in Xixia county, south Yang.

Before the test, the test sheep were subjected to epidemic prevention, anthelmintic, cleaning and disinfection in the test pen, and then 2 groups of test sheep were respectively fed in 2 independent pens. The animals were fed twice daily. All sheep had free water and were free to move.

TABLE 1 basic diet composition (Dry matter basis)

Raw materials%	Control group	Test group
			Ensiling corn	32	32
Peanut vine	8	8
			Sweet potato seedling	8	8
Corn (corn)	27	27
			Wheat bran	6	6
Bean pulp	12	12
			Distiller's dried grain	3	3
Stone powder	1	1
			Calcium hydrogen phosphate	1.13	1.13
Salt	0.87	0.87
			Premix compound①	1	1
Total up to	100	100

Note: the premix provides VA 20000IU and VB for each kilogram of daily ration₅ 25.74mg，VD 30000IU，VE 5000IU，Fe 56mg，Mn 31mg，Zn 92.5mg，Cu 30mg，I 1.25mg，Se 1.00mg。

TABLE 2 Nutrition ingredient levels (Dry matter basis)

Nutritional levels②	Control group	Test group
			Digestive energy/(MJ/kg)	12.24	12.46
Crude protein%	16.96	16.87
			Coarse ash content%	3.49	3.88
Neutral detergent fiber%	37.32	37.47
			Acid detergent fiber%	22.68	22.44
Calcium content%	1.26	1.26
			Phosphorus%	0.54	0.54

Note: ② the digestion energy in each item of the nutrition level is a calculated value, and the other items are measured values.

Example 2 influence of NAM on milk production, milk composition and milk fatty acid content of milk goats

(1) Milk yield

Milking was performed 2 times a day, morning and evening, and milk production data was recorded after each milking.

The milk yield difference between the control group and the test group is not obvious at the beginning of the experiment, and belongs to the difference of milk production traits among normal individuals. As the test is carried out, the milk production of the milk goat is increased from the early lactation period to the full lactation period, and the milk yield of the milk goat is increased at the end of the test compared with that at the beginning of the test. The results show that the milk production of the dairy goats in the group with NAM added daily ration is significantly higher than that in the control group (P <0.05, Table 3). The fact that the Nicotinamide is added into the daily ration can obviously improve the milk yield of the milk goat and can improve the economic benefit of the milk goat.

TABLE 3 influence of NAM on milk production of milk goats

Item	Control group	Test group
			Milk yield (kg/milk) at the beginning of the test (day 1)	2.04±0.14	2.09±0.09
Milk yield (kg/milk) at the end of the test (day 60)	2.32±0.06b	2.48±0.07a

Note: the shoulder marks of the same row indicate that the difference is significant by different lower case letters (P <0.05), the different upper case letters indicate that the difference is significant (P <0.01), and the non-marked letters indicate that the difference is not significant (P > 0.05). The following table is the same.

(2) Milk component

And (3) collecting milk samples for 1 time for all test sheep at the end of the test period, collecting 20mL milk samples respectively in the morning and at the evening to prepare mixed samples for detection, collecting 40 samples of all test sheep, sending the samples to a dairy cow production performance measurement center in Henan province, and analyzing milk component indexes such as lactose rate, milk fat rate, milk protein rate and the like.

As can be seen from Table 4, the milk fat rate of the milk goat milk component added with NAM is significantly higher than that of the control group (P < 0.05); lactose, milk protein and dry matter were not significantly changed (P > 0.05).

TABLE 4 influence of NAM on milk goat milk composition

Item	Control group	Test group	P value
				Milk fat/%)	3.48±0.08b	3.65±0.06a	0.045
Milk protein/%)	3.22±0.09	3.14±0.10	0.347
				Lactose/%)	4.41±0.10	4.29±0.30	0.315
Dry matter/%	12.29±0.15	12.25±0.23	0.798

(3) Milk fatty acid

At the end of the test period, all the test sheep were sampled 1 time with 20mL each in the morning and evening to prepare a mixed sample for fatty acid extraction and determination.

The method for extracting fatty acid from goat milk comprises the following steps: putting the collected milk sample into a water bath kettle at 30 ℃ for incubation for 20 minutes, then centrifuging the milk sample for 30 minutes at 20 ℃ by using a centrifugal force of 17800g, transferring the upper-layer fat into a 2mL centrifuge tube after centrifugation, and centrifuging the milk sample for 20 minutes at 19300 g; taking a 10mL glass tube to absorb 80 μ L of upper-layer oily liquid in the centrifuge tube, adding 5mL of n-hexane, shaking up and mixing; then 0.2mL of methyl esterification reagent (11.2 g of KOH dissolved in 100mL of methanol) is added into the mixture, and the mixture is sealed, vortexed and shaken for 1 minute and then is kept stand for 30 minutes at room temperature; then 0.5g of anhydrous sodium sulfate is added, and 350g of the mixture is centrifuged for 3 minutes; aspirate 1mL of supernatant into a 2mL chromatographic vial for gas chromatography determination. Determining fatty acid components by gas chromatography, and calculating the contents of various fatty acids in the goat milk by an area normalization method.

As can be seen from Table 5, the contents of the fatty acids C4:0, C16:0 and C18:1 in the goat milk were significantly increased (P <0.05) and the contents of the other fatty acids were not significantly changed (P >0.05) in the test group compared to the control group. The result shows that the addition of NAM in the daily ration has the effect of improving the fatty acid composition of the goat milk, and the composition of fatty acid easy to digest and absorb and the content of beneficial fatty acid of the goat milk are improved.

TABLE 5 Effect of daily ration addition of NAM on sheep milk fatty acid composition

Kind of fatty acid	Control group (%)	Test group (%)	P value
				C4:0	1.54±0.23b	1.77±0.21a	0.009
C6:0	1.81±0.23	1.89±0.19	0.274
				C8:0	2.33±0.41	2.32±0.32	0.988
C10:0	9.01±1.51	8.87±1.22	0.794
				C12:0	4.99±0.79	4.70±1.00	0.402
C14:0	10.63±1.50	10.87±1.17	0.649
				C16:0	26.20±2.11b	28.16±2.62a	0.041
C16:1	1.18±0.29	1.11±0.38	0.589
				C18:0	8.20±1.75	8.05±2.16	0.849
C18:1	24.99±1.51b	26.08±1.15a	0.039
				C18:2	2.47±0.47	2.50±0.34	0.850

Example 3 Effect of different concentrations of NAM on SIRT1 and milk fat Synthesis transcription factor expression

In order to research the action mechanism of NAM on the milk fat rate of goat milk and the influence of NAM on lipid metabolism at the cellular level, the mammary epithelial cells of the goat milk were treated with NAM (0,100,300,500. mu. mol/L) at different concentrations.

(1) Cell culture and processing

Separating mammary epithelial cells from mammary tissue of goat by trypsinization, subculturing by high density inoculation, and culturing with DMEM/F12 cell culture medium containing 10% FBS, 100U/mL penicillin, 100 μ g/mL streptomycin, 10ng/mL epidermal growth factor, 5 μ g/mL hydrogenated prednisone and 5 μ g/mL insulin at 37 deg.C and 5% CO₂Culturing for 24h in an incubator, replacing a fresh culture medium, and carrying out subculture when the confluency of the cells reaches over 90% under an inverted microscope. Inoculating the cells into a 12-hole culture plate, culturing until the confluency of the cells reaches 60-70%, respectively adding NAM induction culture media containing 0 [ mu ] mol/L (control), 100 [ mu ] mol/L, 300 [ mu ] mol/L and 500 [ mu ] mol/L, culturing for 48h, collecting the cells, and detecting the expression of milk fat synthesis related genes in subsequent experiments, thereby determining the optimal treatment concentration.

(2) Extraction of total RNA of cell and synthesis of cDNA

And (3) collecting cells after NAM treatment for 48 hours, extracting total RNA of the cells by using a total RNA extraction kit of cell/tissue of Beijing Tiangen Biochemical technology Co. The RNA sample is subjected to ultraviolet spectrophotometer (NANODROP2000) to determine the A260 nm/A280 nm absorption ratio value of the RNA sample, the qualified sample is obtained when the detection concentration is more than 200 ng/mu L and the purity is 1.8-2.0, the extracted RNA is used as a template, and all the RNA which is qualified for detection is subjected to reverse transcription into cDNA according to the steps of a reverse transcription kit of Takara company.

(3) Real-time fluorescent quantitative PCR

The real-time quantitative primers of the milk fat synthetic gene and the reference gene are shown in a table 6, the specific real-time fluorescent quantitative primers are respectively designed by utilizing Primer Premier 5.0 and Oligo 6 software according to the published goat gene sequences in GenBank, a trans-intron method is adopted, the upstream Primer and the downstream Primer of each pair of quantitative primers are distributed on different exons, the length of an amplification product is about 200bp, and each Primer sequence is synthesized by Shanghai biological engineering Co., Ltd.

The experiment performed real-time fluorescent quantitative PCR reactions with the GAPDH and UXT genes as internal references. PCR reaction 20. mu.L: fluorescent quantitative PCR reaction mixture (2 XSSYBR Premix Ex Taq Mix) 10.0. mu.L, cDNA template 1.0. mu.L, upstream and downstream primers (10. mu. mol/L) 0.8. mu.L each, and RNase-free H₂O make up the system. Carrying out reaction on an ABI 7500 fluorescence quantitative PCR instrument under the following reaction conditions: 30s at 95 ℃, 5s at 95 ℃,30 s at 60 ℃ and 40 cycles; a melting curve was added. By using 2^－△△CtThe method analyzes the data, wherein the delta Ct is Ct_{Target gene}-Ct_{Internal reference}；△△Ct＝△Ct_{Test group}-△Ct_{Control group}. 3 replicates were set for each sample.

TABLE 6 real-time quantitative primer sequences

As a result, 300. mu. mol/L of NAM down-regulated the mRNA expression of Sirtuin 2-related enzyme 1(SIRT1) and FoxO1, whereas 100. mu. mol/L of NAM had no effect on SIRT1 and FOXO1 expression and 500. mu. mol/L of NAM had no significant effect on FOXO1 expression (FIG. 1A); NAM at 100. mu. mol/L and 300. mu. mol/L were significantly up-regulated (P <0.05) to PPAR γ, a key regulatory factor for milk fat (FIG. 1B), and 300. mu. mol/L was judged to be the optimum concentration. Cells were treated with 300. mu. mol/L NAM and found to express the fatty acid synthase FASN and the fatty acid desaturase SCD1 in significantly up-regulated amounts (P <0.05) and the lipolytic gene ATGL in significantly down-regulated amounts (P <0.05) (FIG. 2).

Example 4 Effect of NAM on triglyceride and fatty acid Synthesis in mammary cells

(1) Triglyceride (TAG) content determination

Treating mammary epithelial cells of a milk goat with 300 mu mol/L NAM for 48h, digesting with 0.25% trypsin/EDTA mixed solution, collecting cell precipitate, washing the precipitate for 2 times with PBS, transferring the cells to a 1.5mL centrifuge tube, adding 200 mu L PBS into each tube, ultrasonically crushing the cells, detecting the content of triglyceride in the cells according to the operation method given by a cell triglyceride enzyme method detection kit, heating a proper amount of lysate in a 70 ℃ water bath for 10min, centrifuging at room temperature of 2000r/min for 5min, reserving the supernatant for enzymatic determination, reacting 10 mu L of the supernatant with 190 mu L of working solution at 37 ℃ for 10min, and detecting absorbance (D) at the wavelength of 550nm by using a full-automatic enzyme marker_550nm)。

(2) Oil red O dyeing

Treating the cells with NAM for 48h, removing the culture medium, and washing twice with PBS; adding 10% neutral formaldehyde to the culture plate, and fixing the cells for 45 min; adding 1mL of oil red O into each hole of cells, and staining for 30 min; finally, the plate was washed 3 times with PBS and photographed under an inverted microscope.

(3) Fatty acid assay in cells

Culturing mammary epithelial cells in a 60mm cell culture dish, treating with NAM for 48h, removing a culture medium, washing with PBS, digesting with 0.25% trypsin/EDTA mixed solution, collecting cell precipitates, adding 2mL of sulfuric acid/methanol solution with the volume ratio of 0.25%, carrying out ultrasonic treatment for 10min, incubating at 80 ℃ for 1h, cooling to room temperature, adding 2mL of 0.1mol/L hydrochloric acid solution and 800 muL of n-hexane, carrying out vortex oscillation for 30s, centrifuging for 5min at 900g, collecting supernatant to 2mL of siliconized glass tube, adding 0.5g of anhydrous sodium sulfate to absorb excessive water, standing for 12-14 h, carrying out vortex oscillation and centrifugation for 3min at 13800g, absorbing the supernatant, analyzing and determining fatty acid components in the cells by GC-MS (gas chromatography-Mass spectrometer), and taking C19:0 fatty acid as a determination standard substance. The relative proportions of the various fatty acids were calculated as a percentage of the peak area of that fatty acid, with 3 biological replicates per treatment set.

It was found that after nicotinamide treatment of the cells, triglyceride levels in the cells increased significantly (P <0.05) (fig. 3A), oil red O staining showed an increase in lipid droplet accumulation (fig. 3B), the proportion of fatty acids C16:0 and C18:1 in the cells increased significantly (P <0.05, fig. 4B,4D), while the proportion of C14:0 and C18:0 in the cells did not change significantly (fig. 4A, 4C). The results are combined to show that the nicotinamide can promote the synthesis of the mammary epithelial cell lipid of the goat, change the content of beneficial fatty acid in the mammary cells, and provide basic data for further improving the fatty acid composition, the nutritional value and the flavor of the goat milk.

Example 5 SIRT1 mediated NAM transcriptional Regulation of FASN Gene

(1) Cloning and structural analysis of FASN Gene promoter

Previous studies found that NAM alters milk fat synthesis and milk fatty acid composition at the cellular level, and to investigate further the SIRT 1-mediated regulation mechanism of NAM on fatty acid composition in cells, we cloned the FASN promoter, a key enzyme in fatty acid synthesis, and studied the transcriptional regulation mechanism of NAM on FASN.

Obtaining the FASN promoter sequence of the milk goat by using a PCR method. The homology of the FASN gene promoter sequence with cattle, human and mice reaches over 90 percent through the comparison of nucleic acid sequences in NCBI. The core region of FASN promoter has multiple characteristic promoter elements and potential transcription factor binding sites, such as CAAT box, TATAbox, GC-box, E-box, transcription factors AP2, sp1, NF-Y and two binding Sites (SRE) of SREBP1, which are found by prediction analysis of transcription factor online software (FIG. 5). In combination with previous studies, SREBP1 was found to regulate transcription of the FASN promoter.

(2) SIRT 1-mediated influence of NAM on FASN promoter Activity

Cells were treated with SIRT1 inhibitor (NAM) and simultaneously transfected with the FASN promoter luciferase reporter vector, and the FASN promoter activity was detected using the dual luciferase system.

Detecting the luciferase activity: after cells are transfected with FASN promoter recombinant plasmids for 48 hours, the cells are collected, luciferase activity is measured according to a Promega double-reporter gene detection kit, the culture solution is discarded, PBS is used for washing twice, 20 mu L of cell lysate is added to each hole, and the cells are incubated for 20min at room temperature. 20 mu L of lysate is absorbed into a 96-hole white board, 100 mu L of LARII is added, and after rapid and uniform mixing, pGL3 luciferase activity detection (F value) is immediately carried out on a multifunctional microplate luminometer; additional 100. mu.L of 1 XStep & Glo solution was added to detect pRL-TK luciferase activity (R value). The F/R value, i.e., relative luciferase activity, was calculated and used to indicate the relative transcriptional activity of the promoter.

The experimental results show that: FASN promoter activity increased significantly after NAM treatment (P <0.05) (FIG. 6).

(3) SREBP 1-mediated effects of NAM on FASN promoter Activity

In order to research the effect of SREBP1 in SIRT 1-mediated NAM regulation of FASN transcription, site-directed mutagenesis is respectively carried out on SRE1 and SRE2 sites on a FASN promoter, recombinant vector transfected cells are constructed, and the change conditions of FASN promoter activity before and after mutagenesis are detected. Site-directed mutagenesis was performed by overlap extension PCR, and the information of the site-directed mutagenesis primers is shown in Table 7:

TABLE 7 site-directed mutagenesis primers

Note: wherein the italics are the restriction sites and the bold is the mutation sites.

The mutant fragment was ligated to a luciferase reporter vector and sequenced to confirm the success of the mutation (FIG. 7).

As a result, it was found that the FASN promoter activity was significantly decreased after SRE site mutation (P <0.05) (FIG. 8). However, treatment of cells with NAM revealed that NAM significantly increased FASN wild-type promoter activity (P <0.05), while having no effect on SRE1 and SRE2 mutant promoter activity (FIG. 9).

These results can confirm that the SIRT1 mediated NAM regulates the activity of FASN gene promoter of milk goats by regulating SREBP1, and provide theoretical basis for improving goat milk fatty acid composition and goat milk flavor.

The above description is only a preferred embodiment of the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Henan animal husbandry economic school

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<210> 20

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 20

gatacaggct ccactttgat tgc 23

<210> 21

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 21

ccatcgcctg tggagtcac 19

<210> 22

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 22

gtcggataaa tctagcgtag ca 22

<210> 23

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 23

cagcatcttg cctgatttgt a 21

<210> 24

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 24

ctgggcatct aggacatcg 19

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 25

acgccatcga gaaacgctac 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 26

gtgcgcagac tcaggttctc 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 27

tgtggccctt ggatatggtt 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 28

ggttgtcgct gagctctgtg 20

<210> 29

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 29

attacgcgta agaggtgtcc gtgcatagg 29

<210> 30

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 30

cggcgcgccg catgacggca ctgg 24

<210> 31

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 31

cggcgcgccg cataacttca ctgg 24

<210> 32

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 32

cagccaagct gtcagcccat gtggcgtgtc 30

<210> 33

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 33

cagccaagct gtcagtttat gtggcgtgtc 30

<210> 34

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 34

ggaagatctg ggttcccgac tcacaact 28