阅读说明：本技术 一种丙二烯氧化物合成酶、编码基因CsAOS及其应用 (Allene oxide synthetase, coding gene CsAOS and application thereof ) 是由 赵剑 张高阳 崔单单 赵丹丹 于 2020-05-14 设计创作，主要内容包括：本发明属于茶叶加工技术领域,尤其涉及一种丙二烯氧化物合成酶、编码基因CsAOS及其应用。其中丙二烯氧化物合成酶基因CsAOS的核苷酸序列如SEQ ID NO.1所示；丙二烯氧化物合成酶基因CsAOS编码的蛋白的氨基酸序列如SEQ ID NO.2所示。本发明提供了一种来源于茶叶且与茶叶香气相关的丙二烯氧化物合成酶基因CsAOS,且丙二烯氧化物合成酶基因CsAOS在不同适制性红茶绿茶品种中的表达差异显著。本发明通过体外瞬时表达以及体外寡核苷酸反义抑制实验发现并证明了,CsAOS的表达能够显著影响茶叶中挥发性物质以及茉莉酸类物质的含量,为茶叶的加工处理以及培育优良茶树品种提供新的技术手段。(The invention belongs to the technical field of tea processing, and particularly relates to allene oxide synthetase, an encoding gene CsAOS and application thereof. Wherein the nucleotide sequence of the allene oxide synthetase gene CsAOS is shown in SEQ ID NO. 1; the amino acid sequence of the protein coded by the allene oxide synthase gene CsAOS is shown in SEQ ID NO. 2. The invention provides an allene oxide synthetase gene CsAOS which is derived from tea and related to tea aroma, and the allene oxide synthetase gene CsAOS has obvious expression difference in different adaptive black tea and green tea varieties. The invention discovers and proves that the content of volatile substances and jasmonic acid substances in tea can be obviously influenced by the expression of CsAOS through in vitro transient expression and in vitro oligonucleotide antisense inhibition experiments, and provides a new technical means for processing tea and cultivating excellent tea varieties.)

1. An allene oxide synthetase gene CsAOS, the nucleotide sequence of which is shown in SEQ ID NO.1, or a DNA sequence which is hybridized with the DNA sequence limited by SEQ ID NO.1 and encodes the protein with the same function; or DNA molecule which has more than 90% of homology with the DNA sequence limited by SEQ ID NO.1 and codes the same functional protein.

2. An allene oxide synthetase has an amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence which is obtained by substituting and/or deleting and/or adding a plurality of amino acid residues in the sequence shown in SEQ ID NO.2 and has the same protein function; or

Derived from the amino acid sequence shown in SEQ ID NO.2, has more than 98 percent of homology and has the same protein function.

3. Use of the allene oxide synthase gene CsAOS according to claim 1 for the preparation and/or use as an allene oxide synthase.

4. Use of the allene oxide synthase gene CsAOS according to claim 1 for the regulation of volatile substance content in tea.

5. Use according to claim 4, characterized in that: the volatile substances in the tea leaves are at least one of heptanal, hexanal, 2-hexenal, 2, 4-hexadienal, 2, 4-heptadienal, 2-hexen-1-ol acetate, 2-ethylhexanol, benzyl alcohol, linalool, phenethyl alcohol, nonenal, nonanol, geraniol, 3-hexenyl ester hexanoic acid, 3-hexenyl ester or hexanoic acid-hexyl ester.

6. The use of the allene oxide synthase gene CsAOS according to claim 1 for the regulation of jasmonates content and/or salicylic acid content in tea.

7. Use according to claim 6, characterized in that: the jasmonic acid substance is at least one of jasmonic acid, jasmonic acid amino acid and methyl jasmonate.

8. The use of the allene oxide synthase gene CsAOS according to claim 1 for adjusting the content of jasmonic acid precursor OPDA in tea.

9. Use of the allene oxide synthase gene CsAOS according to claim 1 as a marker for screening tea varieties suitable for making green tea and/or tea varieties suitable for making black tea or black tea.

10. A method for promoting the expression level of allene oxide synthase gene CsAOS in tea leaves is characterized in that: comprises at least one treatment mode of withering, rolling and fermenting treatment steps of the tea leaves.

Technical Field

The invention belongs to the technical field of tea processing, and particularly relates to allene oxide synthetase, an encoding gene CsAOS and application thereof.

Background

The black tea is a fully fermented tea, is prepared by using proper tea tree new bud leaves as raw materials through a series of processes of withering, rolling (cutting), fermentation, drying and the like, is a main tea selling class in the international tea market, and is popular with consumers. The polyphenol substances of the tea leaves are subjected to a series of reactions such as oxidation reduction and the like in the fermentation process of the black tea, so that the color of the tea leaves is changed from green to red, and the basic characteristics and the fragrance of the 'red leaf and red soup' are formed, and the black tea has unique taste.

The tea aroma refers to the peculiar smell of tea which is sensed by people because different concentrations and proportions of odor substances influence olfactory nerves. Tea aroma is one of factors playing an important role in tea quality, a plurality of factors influencing tea aroma are provided, such as tea varieties, cultivation conditions, natural environment, processing technology, storage method and the like, and the volatile components in the tea are detected to be hundreds of types and mainly comprise aroma compounds such as aldehydes, ketones, alcohols, esters and the like. The components are combined in different proportions to form tea leaves with different flavors and types. The aroma component substances in the tea leaves are various, but the content of the aroma component substances is very little, the fresh tea leaves account for 0.001-0.05% (dry substances), and the black tea leaves account for 0.01-0.03% (dry substances).

The current research shows that the aroma substances in black tea mainly come from 3 aspects, including terpene compounds such as geraniol, compounds generated by the degradation of carotenoid such as ionone and the like, and oxidized lipid (Oxylipins) compounds generated by the lipid peroxidation of tea cell membranes. In the green leaves of most plants, Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) account for more than 80% of the membrane lipid of the green leaves. During tea processing, the two chloroplast membrane lipids are decomposed under the action of activated phosphatase A1(PLA1) to produce alpha-Linolenic acid (18: 3), which is catalyzed by Lipoxygenase (LOX) to produce 13S-hydroperoxyl Linolenic acid (13-HPOT). 13-HPOT can produce different breakdown products under different physiological conditions or environmental stresses. On one hand, the 13-HPOT is catalyzed and decomposed into C6-C9 volatile substances by lipid hydroperoxide lyase (HPL), wherein the volatile substances comprise 3-hexenal, hexanal and the like with grass fragrance; on the other hand, 13-HPOT is converted into Oxidized Plant Dienoic Acid (OPDA) under the catalysis of Allene Oxide Synthase (AOS) and Allene Oxido Cyclase (AOC) in sequence, the OPDA produced in chloroplasts is transported by transporters into peroxisomes, and Jasmonic Acid (JA) is finally produced via the oxidation of plant dienoic acid reductase (opdaredoctane) and 3 times of β -oxidation. JA is then methylated by JAMT to produce methyl jasmonate. (FIG. 1: synthetic route of jasmonates).

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides allene oxide synthetase, a coding gene CsAOS and application thereof, and aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.

The allene oxide synthetase gene CsAOS has the nucleotide sequence shown in SEQ ID No.1 or the DNA sequence which is hybridized with the DNA sequence limited by SEQ ID No.1 and codes the protein with the same function; or DNA molecule which has more than 90% of homology with the DNA sequence limited by SEQ ID NO.1 and codes the same functional protein.

The invention also discloses an allene oxide synthetase, the amino acid sequence is shown in SEQ ID NO.2, or the sequence shown in SEQ ID NO.2 is subjected to substitution and/or deletion and/or addition of a plurality of amino acid residues, and the allene oxide synthetase has the amino acid sequence with the same protein function; or derived from the amino acid sequence shown in SEQ ID NO.2, has more than 98 percent of homology and has the same protein function.

The invention also discloses application of the allene oxide synthetase gene CsAOS in preparation and/or serving as lipoxygenase.

The invention also discloses application of the allene oxide synthetase gene CsAOS in adjusting the content of volatile substances in tea.

Further, the volatile substances in the tea leaves are at least one of heptanal, hexanal, 2-hexenal, 2, 4-hexadienal, 2, 4-heptadienal, 2-hexen-1-ol acetate, 2-ethylhexanol, benzyl alcohol, linalool, phenethyl alcohol, nonenal, nonanol, geraniol, 3-hexenyl ester hexanoic acid, 3-hexenyl ester or hexyl ester hexanoate.

The invention also discloses application of the allene oxide synthetase gene CsAOS in adjusting the content of jasmonic acid substances and/or salicylic acid in tea.

Further, the jasmonate substance is at least one of jasmonic acid, jasmonic amino acid and methyl jasmonate.

The invention also discloses application of the allene oxide synthetase gene CsAOS in adjusting the content of jasmonic acid precursor OPDA in tea.

The invention also discloses application of the allene oxide synthase gene CsAOS in screening tea varieties suitable for making green tea and/or tea varieties suitable for making black tea or black tea as a marker.

The invention also discloses application of the allene oxide synthase gene CsAOS in tea tree breeding.

The invention also discloses a method for promoting the expression of the allene oxide synthetase gene CsAOS in the tea, which comprises at least one treatment mode of withering, rolling and fermentation treatment steps of the tea.

In summary, the advantages and positive effects of the invention are:

the invention provides an allene oxide synthetase gene CsAOS derived from tea leaves and related to tea leaf aroma, and a protein coded by the allene oxide synthetase gene CsAOS has the function of an allene oxide synthetase.

According to the invention, the expression difference of the allene oxide synthase gene CsAOS in the black tea processing process is detected, so that the expression of the gene is promoted by the withering, rolling and fermentation in the black tea processing process, and the gene is shown to participate in the black tea making process.

The invention discovers and proves that the content of volatile substances and jasmonic acid substances in tea can be obviously influenced by the expression of CsAOS through in vitro transient expression and in vitro oligonucleotide antisense inhibition experiments, and provides a new technical means for processing tea and cultivating excellent tea varieties.

Drawings

FIG. 1 is a synthetic pathway for jasmonates;

FIG. 2 shows the expression difference of allene oxide synthase gene CsAOS in different tissues of Shuchazao according to an embodiment of the present invention; wherein, L1 is a leaf; l2 two blades; l3, trefoil; FL is flower; FR is fruit; s, stem; r is root; b, bud;

FIG. 3 shows the difference in expression of allene oxide synthase gene CsAOS during black tea processing according to an example of the present invention;

FIG. 4 is the expression of CsAOS in different adapted tea varieties according to an embodiment of the present invention;

FIG. 5 shows the expression level of allene oxide synthase gene CsAOS in an in vitro oligonucleotide antisense suppression experiment according to an embodiment of the present invention;

FIG. 6 shows the results of jasmonates assay 3d after oligonucleotide antisense suppression experiment in vitro according to an embodiment of the invention;

FIG. 7 shows the determination of volatile substances after 3d of antisense inhibition experiment of oligonucleotides in vitro according to an embodiment of the present invention;

FIG. 8 shows the expression level of the allene oxide synthase gene CsAOS transiently expressed in vitro according to the embodiment of the present invention;

FIG. 9 shows the results of jasmonates assay after 3d in vitro transient expression according to an embodiment of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

The invention discloses an allene oxide synthase gene CsAOS, and a coding protein and application thereof, which are shown in the following embodiments. The nucleotide sequence of the allene oxide synthetase gene CsAOS is shown as SEQ ID NO.1 in a sequence table, and the amino acid sequence is shown as SEQ ID NO.2 in the sequence table.

The materials involved in the invention:

1. tea tree samples: the Shucha early tea tree is planted in the agricultural industrial park of Yihui agricultural university of Yihui, Luyang area of Anhui province, and the leaf picking condition is 25-28 ℃.

The preparation process of the black tea mainly comprises the steps of picking, withering, rolling, fermenting and drying. The method specifically comprises the following steps: picking one bud and one leaf of Shucha early plant (in this case, a fresh leaf sample); spreading at room temperature for about 16h (withering sample after completion); firstly, lightly kneading tea leaves into a spherical shape, then heavily kneading and lightly kneading (a kneading sample is obtained after the completion); wrapping the tea leaves with gauze, fermenting at 30 deg.C for 12h (fermentation sample after completion), dispersing every 2h during fermentation period, and continuing fermentation; drying at 110 ℃ for about 15min (drying samples after completion).

Tobacco sample: the Nicotiana benthamiana (Nicotiana benthamiana) is grown in a light culture chamber at a temperature of 22-25 ℃.

2. Coli: DH5 α.

3. Carrier: pGEM-T Easy.

4. LB culture medium: weighing 10g of NaCl, 5g of yeast extract and 10g of tryptone, adding 950mL of ultrapure water, stirring and dissolving, adding water to a constant volume of 1000mL, and sterilizing for 15min by high-pressure steam to obtain an LB liquid culture medium, wherein the LB solid culture medium is obtained by adding 15g of agar powder into the LB liquid culture medium.

5. Ampicillin mother liquor (Amp +, 50 mg/ml): 0.5g ampicillin Amp was weighed, dissolved in 10mL sterile water, filtered, sterilized, and dispensed into vials for storage at-20 ℃.

6. In vitro oligonucleotide antisense inhibition buffer: centrifuging nucleotide powder (8000rpm,5min), adding 2ml of 50mMol/L sucrose solution into each tube, shaking on a vortex apparatus for several minutes, observing whether the powder is completely dissolved, stopping shaking if no particulate matter is found, obtaining in-vitro oligonucleotide antisense inhibition buffer solution (hereinafter referred to as buffer solution), transferring 250ul of buffer solution by using a liquid transfer gun, and subpackaging in a 96-well plate for later use.