CN106701819B

CN106701819B - Method for increasing content of polyamine metabolism-related products of brown rice and rice

Info

Publication number: CN106701819B
Application number: CN201710131667.9A
Authority: CN
Inventors: 李建粤; 赵国超; 谢米雪; 张涛
Original assignee: Shanghai Normal University
Current assignee: Shanghai Normal University
Priority date: 2017-03-07
Filing date: 2017-03-07
Publication date: 2021-06-25
Anticipated expiration: 2037-03-07
Also published as: CN106701819A

Abstract

The invention relates to the technical field of agriculture, and discloses a method for increasing GABA content in brown rice, which aims to increase GABA content in brown rice by increasing the content of polyamine substances, which are related metabolites of a polyamine metabolic pathway of rice. Can enhance the expression of related genes in polyamine metabolic pathway in the rice seed brown rice, such as arginine decarboxylase gene, ornithine decarboxylase gene, polyamine oxidase gene or diamine oxidase gene; or stress treating the rice to induce the increase of the content of polyamine-related metabolites in the brown rice of the rice. The method improves the GABA content in the brown rice to a greater extent, is convenient to operate and has obvious effect. Can be used for normal embryo rice brown rice and giant embryo rice brown rice.

Description

Method for increasing content of polyamine metabolism-related products of brown rice and rice

Technical Field

The invention relates to the technical field of agriculture, in particular to a novel method for improving GABA content of brown rice of rice.

Background

Gamma-aminobutyric acid, GABA for short, is a non-protein functional amino acid. GABA has very important health care function to human body. It has been reported that rice germs rich in GABA are orally administered to improve insomnia, depression and vegetative nerve dysfunction symptoms in women with climacteric syndrome (Okada et al, J.Jpn.Soc.food Sci.,2000,47(8): 596-. A report by the American scholar, Leventhal et al, published in 2003 in the world's famous journal, Science, demonstrated that GABA improves visual cortex function in senescent monkeys (Leventhal et al, Science,2003,300(5620): 812-. Further studies by Hua et al found that in the visual cortex of senior cats, the neuron density and proportion containing GABA were significantly less than in young adult cats. They speculate that it is due to low levels of GABA transmitters in older animals that the visual function is degraded (Hua et al, Brain Research Bulletin,2008,75: 119-. Studies by Ge et al suggest that GABA plays an important role in synaptic potential integration of newly formed neurons in the adult brain (Ge et al, Nature,2006,439(7076): 589-. The study of the tulbaghia, et al found that the GABA content in the serum of the cerebral palsy infant is significantly lower than that of the control group (P < 0.05). They believe that GABA is involved in the pathophysiological processes of cerebral palsy, and GABA is a biochemical indicator reflecting the degree of brain injury and may be involved in the pathogenesis of cerebral palsy (coquette et al, chinese medicine for disability, 2007,15(2): 2-4). There are also reports of studies to date which demonstrate that when GABA-rich rice is fed to hypertensive and normotensive mice, it is found that after six weeks, the blood pressure in hypertensive mice is reduced by 20 mm Hg compared to the original blood pressure in normal mice, while the blood pressure in normal mice is not affected (Akama et al, Transgenic research,2009,18(6): 865-. Recent literature reports have shown that GABA induces the transformation of alpha islet cells into beta islet cells capable of producing insulin, and has new promise for the treatment of diabetes (Ben-Othman et al, Cell,2017,168: 1-13). In the review of ' physiological function and research and development progress of gamma-aminobutyric acid ' written by scholars in China ' Yangyuan and the like, GABA is also mentioned to be capable of reducing blood pressure, promoting sleep, enhancing memory and resisting anxiety, and the deficiency of GABA is also related to the occurrence of nervous system diseases such as cerebral senility, epilepsy and the like and the abnormal reproduction phenomenon (Yangyuan and the like, food science, 2005,26(9): 546-. It is also mentioned in centuries about the physiological effects of GABA that the decrease in GABA in nervous tissues is also associated with the development of neurosis such as senile dementia; GABA can promote ethanol metabolism and relieve hangover; GABA also has the effect of preventing skin aging (http:/Baike. ***. com/view/405772. htm).

GABA is widely present in animals and plants, but accumulation of GABA becomes difficult as the organism ages and mental stress increases. Therefore, in daily diet, the proper supplement of GABA is beneficial to human health.

Rice is one of the most important grain crops in the world and China, and is at least staple food on 1/2 population dining tables in China. At present, with the improvement of living standard of people, people pay more and more attention to and pay more attention to self health problems. People pay more attention to how to eat well and eat healthily at the same time of being full. If people can supplement GABA when eating rice at ordinary times, the rice is beneficial to the health of people.

In recent years, with the increase of the incidence of various cardiovascular diseases such as hypertension and diabetes, the research on how to increase the GABA content of the brown rice has been more and more focused.

In 1994 early, Saikusa et al, Japan scholars reported that the composition of free amino acids changed after soaking general normal embryo rice brown rice in water, with the accumulation of gamma-aminobutyric acid (GABA) being the most significant (Saikusa et al, biosci. Biotech. biochem.,1994,58(12): 2291-2292). Zhanglin et al believe that GABA content in brown rice increases by embryo size (Zhanglin et al, nuclear agriculture bulletin 20016,20(3): 218-. Therefore, the GABA content of brown rice can be increased by growing giant embryo rice (Seo et al, Journal of the Korea Society for Applied Biological Chemistry,2011,54(5): 700-. Liu et al (2005) found that GABA content increased significantly after soaking giant embryo brown rice in water. Liu et al (2005) were further investigated and suggested to be primarily due to the massive synthesis of GABA from glutamic acid decarboxylase (Liu et al, cellular Chem.,2005,82(2): 191-196).

A current report on the accumulation of GABA in plants has recognized that it is synthesized from the decarboxylation of glutamic acid (Glu) by glutamate decarboxylase (GAD, EC4.1.1.15) (Choi et al, J.biochem.mol.biol.,1998,31: 233-239; Park et al, J.biochem.mol.biol.,1999,32: 339-344). In 2007, Akama et al expressed OsGAD2 cDNA in glutamate decarboxylase isogene, and cDNA fragments remaining after cutting 31 amino acids from C-terminus: GAD2 Δ C, and expression of both fragments was then achieved in E.coli. Then the two fragments are respectively connected with a cauliflower mosaic virus 35S gene promoter and transformed into rice, and the GABA content in the callus after transformation is detected, so that the GABA content of the callus of the two transgenic rice is obviously higher than that of the non-transgenic rice (Akama and Takaiwa, J Exp Bot,2007,58: 2699-one 2707). On the basis of 2007 research, Akama and the like connect a GAD2 delta C fragment with a glutelin GluB-1 gene promoter specifically expressed by rice endosperm to construct an expression vector, and transform normal embryo rice to obtain Transgenic offspring, and the result shows that GABA content in Transgenic rice grains is remarkably improved (Akama and the like, Transgenic research,2009,18(6): 865-.

The prior art does not fully reveal the mechanism of GABA accumulation in brown rice, whether it is also accomplished by decarboxylation of glutamate by glutamate decarboxylase? Whether the molecular mechanism of further increasing GABA content during the soaking of normal embryo brown rice and giant embryo brown rice is thought to be due to the fact that glutamic acid is synthesized in large amount under the action of glutamate decarboxylase?

The invention discloses a way for generating GABA through a polyamine metabolic pathway in rice so as to establish a novel method capable of improving GABA content of brown rice of rice.

Disclosure of Invention

The invention aims to provide a method for improving GABA content in brown rice.

The invention establishes a new method for improving GABA content of brown rice, and the scheme is to increase the GABA content of the brown rice by increasing the content of related metabolites in a polyamine metabolic pathway of the rice.

The related metabolites of the polyamine metabolic pathway are polyamine related substances, including at least one of putrescine, spermidine, spermine, arginine and ornithine. The polyamine substances in plants are mainly putrescine, spermidine and spermine, and the putrescine content is generally the highest among the three. The related metabolites in the polyamine metabolic pathway mainly include putrescine, spermidine, spermine and related precursors arginine, ornithine and the like.

There are various methods for increasing metabolites related to polyamine metabolic pathway (polyamine-related substances). The method comprises the following steps:

(1) enhancing the expression of related genes in polyamine metabolic pathways in the rice seeds and brown rice; alternatively, the first and second electrodes may be,

(2) the rice is subjected to stress treatment to induce the increase of the content of polyamine-related metabolites in the brown rice of the rice.

The stress processing method comprises the following steps: drought treatment, soaking for accelerating germination or reducing the environmental temperature of rice seeds in the mature period. For example, rice plants are subjected to drought treatment by changing the planting environment of rice, particularly in the late stage of rice planting; alternatively, the increase in the content of polyamine-related metabolites in the brown rice of rice can be induced by changing the environmental temperature, particularly by lowering the environmental temperature during the mature period of rice seeds. In a preferred embodiment of the invention, the environmental temperature of the rice seed in the mature period is 15-21 ℃, so that the GABA content in the brown rice can be obviously increased.

The related gene in the polyamine metabolism pathway is at least one of arginine decarboxylase gene, ornithine decarboxylase gene, polyamine oxidase gene or diamine oxidase gene. The arginine decarboxylase gene is at least one of OsADC1, OsADC2 or OsADC3 genes; the ornithine decarboxylase gene is at least one of OsODC1, OsODC2 and OsODC3 genes; the polyamine oxidase is at least one of OsPAO2, OsPAO6 and OsPAO7 genes; diamine oxidase is an OsDAO gene.

The expression of related genes in a polyamine metabolic pathway in the brown rice of the rice seed is enhanced, a regulatory gene or a structural gene capable of enhancing the synthesis of various related metabolites in the polyamine metabolic pathway is introduced into the rice seed, and the transgenic rice with the increased content of the polyamine related metabolites in the brown rice of the rice seed is cultivated by a method of transferring an exogenous gene.

The rice is common normal embryo rice or giant embryo rice. For example, "super 2-10" normal embryo rice or "Shanghai Large No. 5" giant embryo rice. The effect is more obvious in giant embryo rice.

The new method for improving GABA content of the brown rice can be used for improving GABA content of the brown rice with normal embryo, can also be used for improving GABA content of the brown rice with giant embryo, or is used on the basis that GABA content of the brown rice is improved by introducing GAD2 delta C fragments, so that GABA content of the brown rice is expected to be further increased to a greater extent.

The invention discloses a way for generating GABA through a polyamine metabolic pathway in rice and establishes a method for improving GABA content of brown rice of rice. The method can improve GABA content in the brown rice to a greater extent, and has convenient operation and obvious effect. The method can be used for normal embryo rice brown rice and giant embryo rice brown rice.

Drawings

FIG. 1 is a graph showing the analysis of the expression of three glutamate decarboxylase genes during the development of two test rice seeds in example 1.

FIG. 2 is a graph showing the analysis of the expression of three arginine decarboxylase genes during the development of two rice seeds tested in example 1.

FIG. 3 is a graph showing the analysis of the expression of three ornithine decarboxylase genes during the development of two rice seeds tested in example 1.

FIG. 4 is a graph showing the analysis of the expression of three polyamine oxidase genes during the development of two test rice seeds in example 1.

FIG. 5 is a graph showing the analysis of the expression of diamine oxidase genes during the development of two test rice seeds in example 1.

In FIGS. 1 to 5, the Y-axis represents the Relative expression level of the gene (Relative expression lever), and the X-axis represents the number of days spent on rice (Day after poling). Indicates that the relative expression amount of the two test paddy rice has a very significant difference P (< 0.01).

FIG. 6 is the analysis of the expression of key enzyme genes in the glutamic acid metabolic pathway and polyamine metabolic pathway after germination of two test rice seeds in example 3. Wherein A is three glutamate decarboxylase (GAD) gene analysis; b is three Arginine Decarboxylase (ADC) gene analyses; c is three Ornithine Decarboxylase (ODC) gene analysis; d is analysis of diamine oxidase (DAO) gene and three polyamine oxidase (PAO) genes. The Y-axis represents the Relative expression level (Relative expression level) of the gene, and the X-axis represents the corresponding gene type. Indicates that the relative expression amount of the two test paddy rice has a very significant difference P (< 0.01).

Detailed Description

Example 1 molecular mechanism for GABA content accumulation in Rice seed development

Arginine (Arg) can be used to synthesize putrescine by Arginine Decarboxylase (ADC). Ornithine may be decarboxylated by Ornithine Decarboxylase (ODC) to synthesize putrescine. Polyamine oxidase (PAO) catalyzes the reverse synthesis of spermidine and spermine to form putrescine. Diamine oxidase (DAO) is a key enzyme in the production of GABA from putrescine.

Arginine Decarboxylase (ADC), Ornithine Decarboxylase (ODC), polyamine oxidase (PAO), and diamine oxidase (DAO) are respectively among some important enzyme proteins in the polyamine metabolic pathway. These enzyme proteins also each have some isoenzyme members.

The normal embryo rice 'super 2-10' and the huge embryo rice 'Shangshda No. 5' are taken as test materials and cultivated and planted according to a conventional method. At 6 days, 10 days, 14 days, 18 days, 22 days, 26 days and 30 days after the rice blossoms, caryopsis (immature brown rice) of young seeds are taken, and a quantitative PCR method is adopted to carry out quantitative PCR on key enzyme genes in a polyamine metabolic pathway: different isozyme gene members of OsADC, OsODC, OsPAO: expression level analysis was performed for OsADC1, OsADC2, OsADC3, OsODC1, OsODC2, OsODC3, OsPAO2, OsPAO6, OsPAO7, and OsDAO genes.

The decarboxylation of glutamate by glutamate decarboxylase (GAD) forms GABA. In example 1, three isozyme gene members of glutamate decarboxylase (GAD), which are currently recognized to play a role in GABA accumulation in rice seeds, were also selected simultaneously: OsGAD1, OsGAD2 and OsGAD3 were used together to analyze gene expression level.

In general, in two rice caryopses, three glutamate decarboxylase genes OsGAD1, OsGAD2 and OsGAD3 have higher expression levels in different periods of seed development, but in the later period of seed development, the expression levels of the three glutamate decarboxylase genes in the giant embryo rice of Shang teacher and 5 are all lower than that of the normal embryo rice of 'super 2-10' (FIG. 1).

In the super 2-10 rice, the different members of the Arginine Decarboxylase (ADC) gene and Ornithine Decarboxylase (ODC) gene, OsADC1, OsADC2, OsADC3, OsODC1, OsODC2, and OsODC3, were very low in the rice seed development process, while they were relatively high in the "shang shi da No. 5" rice (fig. 2 and fig. 3).

Three gene members of a polyamine oxidase (PAO) gene, namely OsPAO2, OsPAO6 and OsPAO7, have very low expression level in the early development stage of 'super 2-10' rice seeds, and the expression levels of OsPAO2 and OsPAO7 are improved in the late development stage of the seeds compared with the early development stage. However, in the giant embryo rice "Shanghai 5", the expression levels of OsPAO2, OsPAO6 and OsPAO7 were all higher than those of "super 2-10" rice (FIG. 4).

Diamine oxidase (DAO) is a key enzyme in the metabolic pathway from putrescine to GABA. In the "super 2-10" rice, the expression level of the OsDAO gene was also lower than that of "Shangsha No. 5" giant embryo rice (FIG. 5) at each development stage.

Selecting caryopses of rice at 26 days and 30 days of flowering, utilizing an amino acid analyzer to analyze the contents of glutamic acid (Glu) serving as a substrate of glutamic acid decarboxylase and arginine (Arg) and ornithine (Orn) in a polyamine metabolic pathway, and simultaneously detecting the content of gamma-aminobutyric acid (GABA). The putrescine, spermidine and spermine contents of the caryopsis during the 26 th and 30 th day of flowering were determined by HPLC method.

The GABA content of the rice caryopsis of Shangda No. 5 is about 60mg/100g at 26 days and 30 days after flowering, and is 2 times of that of the rice of super 2-10 at the same time. The contents of arginine and ornithine which are used as substrates for synthesizing putrescine are also remarkably higher than those of 'super 2-10' rice in 'Shangdao 5' rice. In particular, arginine is particularly remarkably improved in the rice seeds of Shanghai Da No. 5, which is about 5 times of that of 'super 2-10' rice. Glutamate was detected in the caryopsis of both rice stages, but their content was very significant or significantly lower for "Master No. 5" than for "super 2-10" rice (tables 1 and 2).

From the results of quantitative analysis of three polyamine substances in caryopsis 26 days and 30 days after blooming in two kinds of test rice, the putrescine content of "shangda 5" is significantly greater than that of "super 2-10" rice, and is shown to be increased by about 2 times (tables 3 and 4).

TABLE 1 amino acid content 26 days after flowering of two test rice species

Note that data are expressed as mean ± standard deviation (n ═ 3). The same applies below.

TABLE 2 amino acid content 30 days after flowering of two test rice species

TABLE 3 polyamine content 26 days after flowering of two test rice species

TABLE 4 polyamine content 30 days after flowering of two test rice species

Giant embryo rice "Shangshanda No. 5" is cultured by single gene (giant embryo gene) mutation in the process of in vitro culture of mature embryo of normal embryo rice "super 2-10" (Wuyonggang et al, Shanghai university report (Nature science edition), 2011,40(3):289 and 294). The "Shanghai 5 and" super 2-10 "have the same genetic background except the difference of genes affecting the size of embryo volume. As known, in the 'Shangda No. 5' giant embryo rice brown rice, the GABA content is higher than that of the normal embryo rice by 'over 2-10', so that the molecular mechanism of GABA accumulation in the rice seed development process can be more clearly proved by using the two rice varieties as test materials for comparative research.

Expression analysis of glume glutamate decarboxylase genes and related enzyme genes in polyamine metabolism pathways 26 days and 30 days after flowering of two types of rice and analysis results of related substances in the two metabolism pathways show that the rice seeds have functions of accumulating the GABA content of the brown rice in the development process, the glutamic acid pathway and the polyamine pathway, and particularly, the contribution to the accumulation of the GABA content of the brown rice of the giant embryo rice is not the function of the glutamic acid pathway but the function of the polyamine metabolism pathway.

Example 2 accumulation of GABA content in brown rice of rice enhanced by polyamine substance content change caused by environmental change

Yang et al (Yang et al, J.Exp.Bot.,2007, 58(6):1545-1555) found that polyamine substance content in rice leaves accumulated under drought conditions. It has been reported that polyamines accumulate mostly in adverse environments. Polyamines have also been associated with plants responding to environmental stresses, including mineral deficiencies, osmotic pressure and drought, salt and alkali tolerance, low temperatures, hypoxia, environmental pollution, etc. (Groppa et al, Amino acids, 2008,34: 35-45; Alcazar et al, Planta,2010,231: 1237-.

This example is to explore whether the factor affecting the GABA content in brown rice is the currently recognized role of the glutamic acid pathway or is related to the polyamine metabolism pathway when the GABA content of two kinds of rice planted in different years is different.

The normal embryo rice 'super 2-10' and the giant embryo rice 'Shangshandao No. 5' are taken as test materials and are planted in 5-10 months in 2013 and 5-10 months in 2014 respectively. The two kinds of rice are completely the same in field block, planting mode, fertilizer and water management, etc. However, when the temperature of the mature stage of the seed development is found by comparing two types of rice flowering and filling in two years, namely, the days from 9 month and 1 to 10 month and 25, the maximum temperature variation range is 18 ℃ to 34 ℃ and the minimum temperature variation range is 12 ℃ to 26 ℃ in the days from 9 month and 1 to 10 month and 25 in 2013. The variation range of the highest temperature in the same period of 2014 is 21-31 ℃, and the variation range of the lowest temperature is 13-23 ℃. Comparing the temperatures of 2013 and 2014 in the mature period of the two rice seeds, the temperature change range is wide in 2013, the temperature exceeds 30 ℃ in 14 days in 9 months, and the temperature exceeds 30 ℃ in 9 months in 2014 for only 5 days. Comparing the temperatures of the positive rice seed maturity periods between 10 months and 15 days to 25 days in the two years, the temperatures in 11 days in 2013 are lower than the temperatures in the same period in 2014, and the temperatures in 5 days are lower than the temperatures in the same period in 2014 by 5 ℃ and more. The average maximum temperature was 21 ℃ and the average minimum temperature was 15 ℃ over 11 days in 2013, while the average maximum temperature was 25 ℃ and the average minimum temperature was 17 ℃ over 11 days in 2014.

And taking the brown rice after the seeds are ripe, detecting the amino acid content and GABA content of the brown rice by adopting an amino acid analyzer, and analyzing the three polyamine contents of the brown rice by adopting an HPLC method. The results are shown in Table 5. When the same kind of rice is planted in different years, the GABA content in the brown rice is different. The GABA content of the two kinds of rice brown rice, namely 'super 2-10' and 'Shangshao 5', is higher in the brown rice harvested in 2013 than in 2014. Compared with the brown rice harvested in 2014, the two kinds of brown rice harvested in 2013 have obviously improved arginine content, ornithine content, putrescine content and spermidine content, particularly the three kinds of polyamine substances are remarkably increased, and the glutamic acid content is reduced. Therefore, the rice planted in different years promotes the increase of the metabolite content in the polyamine general way due to the difference of the growing environment temperature (the environment temperature is higher in the early filling stage of rice in 2013, but is relatively lower in the mature period of seeds), thereby promoting the synthesis of GABA in the brown rice.

The results of this example show that the factors that influence the accumulation of GABA content in brown rice are related to polyamine metabolism pathway. Therefore, the GABA content in the brown rice can be increased by adjusting the temperature of the rice planting environment.

TABLE 5 detection of amino acid content and polyamine content in brown rice of two kinds of rice under different planting environment temperatures

EXAMPLE 3 Brown Rice soaking treatment to increase GABA content

The normal embryo rice 'super 2-10' and the giant embryo rice 'Shangshda No. 5' are taken as test materials. Soaking the brown rice in clear water at 25 deg.C for 48 hr, replacing the clear water every 12 hr, draining water after 48 hr, and germinating at 28 deg.C for 12 hr. Freezing part of the germinated brown rice at-80 deg.C for GABA metabolic pathway key enzyme gene expression analysis, sun drying part of the germinated brown rice, oven drying at 50 deg.C, and storing in refrigerator at 4 deg.C for amino acid content and GABA content and three polyamine content analysis by using amino acid analyzer and HPLC.

The expression level of glutamate decarboxylase gene OsGAD2 was very low in both of the test rice plants, but the other two glutamate decarboxylase genes OsGAD1 and OsGAD3 were expressed in both of the rice germinated brown rice plants, but their expression levels were opposite in both of the rice plants (FIG. 6A).

The expression levels of OsADC1 and OsADC3 in the arginine decarboxylase gene were also very low in both of the two test rice plants, and the expression level of OsADC2 was higher in both of the two test rice plants, but the expression level of OsADC2 gene was higher in the normal embryo rice "super 2-10" germinated brown rice than in the giant embryo rice "Shang teacher No. 5" germinated brown rice (FIG. 6B).

The expression level of ornithine decarboxylase OsODC1 gene in two kinds of rice germinated brown rice is higher than that of the other two kinds of rice germinated brown rice genes OsODC2 and OsODC 3. The expression levels of the three ornithine decarboxylase genes in the two types of rice germinated brown rice were compared, and were higher in the "shang teacher large 5" germinated brown rice than in the "super 2-10" germinated brown rice (fig. 6C).

The expression level of the OsDAO gene was higher in both the two types of germinated brown rice, but the comparison of the two types of rice revealed that the expression level of the OsDAO gene in the "Shangsha No. 5" germinated brown rice was significantly higher than that of "super 2-10" (FIG. 6D).

The expression levels of OsPAO2 and OsPAO7 in the three polyamine oxidase (PAO) genes in the two types of germinated brown rice are higher than that of OsPAO6, and the expression levels of OsPAO2 and OsPAO7 in the 'super 2-10' germinated brown rice are higher than that of 'Shangshan 5' rice (FIG. 6D).

Comparing the amino acid and polyamine metabolite contents of the two kinds of rice brown rice before and after germination, the contents of other substances are obviously increased after germination except that the spermine content in the seeds after germination is less than that before germination. The extent of putrescine increase was most pronounced in both rice plants compared before and after germination (table 6).

TABLE 6 detection of amino acid content and polyamine content of brown rice before and after germination of two kinds of rice

The results of analysis of expression of related genes in the glutamic acid metabolic pathway and the polyamine metabolic pathway after germination of two kinds of rice and analysis of related substance contents of the two kinds of rice before and after germination show that the accumulation of GABA content in rice brown rice germination is related to not only the glutamic acid metabolic pathway but also the polyamine metabolic pathway.

Example 4 promoting the content of polyamine-related metabolites to increase the GABA accumulation of brown rice by controlling the gene expression level of an enzyme protein of a polyamine biosynthesis pathway through transgenic operation

Structural genes capable of enhancing synthesis of various related metabolites in a polyamine metabolic pathway, such as three members OsADC1, OsADC2 and OsADC3 in an Arginine Decarboxylase (ADC) gene, or three members OsODC1, OsODC2 and OsODC3 in an Ornithine Decarboxylase (ODC) gene, or three members OsPAO2, OsPAO6 and OsPAO7 in a polyamine oxidase (PAO) gene are used as target genes, and promoters capable of being expressed in brown rice, such as promoters of various rice gluten genes, rice globulin genes, rice waxy genes and the like, are selected to construct an overexpression vector and are introduced into the rice to obtain transgenic rice containing the exogenous genes. Then obtaining homozygous transgenic plants according to the conventional operation. The culture of Agrobacterium tumefaciens, transformation of rice and selection of resistant callus and plant regeneration and screening of homozygous material involved in the cultivation of transgenic rice are mainly carried out by referring to the method of Li Jianyue et al (Li Jianyue et al, journal of northwest plant, 2008, 28: 1082-.

Marking when the transgenic rice blooms, taking caryopses at the 30 th day after blossoming to carry out the expression analysis of the target gene of the transgenic rice, and harvesting seeds after the rice is mature to carry out GABA content detection. The OsADC1, OsADC2 and OsADC3 genes have lower expression levels in non-transgenic rice caryopsis, and particularly the OsADC3 gene hardly expresses. For caryopsis of transgenic rice which is transformed with three OsADC1, OsADC2 and OsADC3 genes together, as long as the relative expression quantity of the OsADC1 gene is compared with that of the OsADC1 gene of non-transgenic rice, the ratio of the relative expression quantity of the OsADC1 gene to that of the non-transgenic rice exceeds 100, the GABA content in mature transgenic rice seeds is increased by 100 percent compared with that of the non-transgenic rice.

By the same operation as above, three members, OsODC1, OsODC2 and OsODC3, which simultaneously contained three Ornithine Decarboxylase (ODC) genes were obtained. And marking when the transgenic rice blooms, taking caryopses at the 30 th day after blossoming to perform target gene expression analysis on the transgenic rice, and harvesting seeds after the rice is mature to perform GABA content detection. The expression levels of the OsODC1, OsODC2 and OsODC3 genes in the caryopsis of the non-transgenic rice are also lower. For caryopses of transgenic rice transformed with three OsODC1, OsODC2 and OsODC3 genes together, as long as the relative expression quantity of the OsODC1 gene is compared with that of the OsODC1 gene of non-transgenic rice, the ratio of the relative expression quantity of the OsODC1 gene to the relative expression quantity of the OsODC1 gene of the non-transgenic rice exceeds 100, the GABA content in mature transgenic rice seeds is at least 50% higher than that of the non-transgenic rice.

This example mainly takes Arginine Decarboxylase (ADC) gene and Ornithine Decarboxylase (ODC) gene in the structural genes as examples to illustrate how to use transgenic technology to increase GABA content in brown rice.

The GABA content of the brown rice can be increased by cultivating the transgenic rice with increased polyamine-related metabolite content in the brown rice according to the same operation by replacing the target gene with other structural genes capable of enhancing the polyamine metabolic pathway or replacing the target gene with a regulatory gene capable of enhancing the polyamine metabolic pathway.

Example 5 additional increase in GABA accumulation in brown rice based on giant embryo rice with higher GABA content

Reports show that the giant embryo brown rice contains higher GABA content, a regulatory gene or a structural gene which can enhance the synthesis of various related metabolites in a polyamine metabolic pathway, such as an Arginine Decarboxylase (ADC) gene, an Ornithine Decarboxylase (ODC) gene and a polyamine oxidase (PAO) gene, is used as a target gene, and meanwhile, a promoter capable of being expressed in the brown rice, such as promoters of various gluten genes, or promoters of globulin genes, or promoters of waxy genes, and the like, is used for constructing an overexpression vector, is introduced into rice seeds, and is used for cultivating transgenic rice with the increased polyamine related metabolite content in the giant embryo brown rice, so that the GABA content of the giant embryo brown rice is further increased.

Claims

1. A method for increasing the content of polyamine-related metabolites in brown rice of rice is characterized by comprising the following steps:

soaking rice for accelerating germination, and inducing the increase of the content of polyamine-related metabolites in brown rice of the rice, wherein the polyamine-related metabolites comprise at least one of putrescine, spermidine, arginine and ornithine; the rice is No. 5 or 2-10 of Shangdao.