AU2021103812A4 - Rice Flowering-related Gene LHD3 and Its Application - Google Patents
Rice Flowering-related Gene LHD3 and Its Application Download PDFInfo
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- C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
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Abstract
The invention discloses a rice flowering-related protein, an encoding gene LHD3 thereof and
an application thereof in rice breeding. The invention provides a rice flowering-related protein,
and the amino acid sequence thereof is shown as SEQ ID NO: 2. The invention also provides
a gene LHD3 encoding the protein. The nucleotide sequence of the gene LHD3 is shown as
SEQ ID NO: 1. The application of the gene LHD3 in improving heading date and
photomorphogenesis of rice. According to the invention, the rice flowering gene LHD3 is
isolated, cloned and identified. The gene function is also verified through a complementary
experiment. The results of map-based cloning showed that the gene encoded rice phytochrome
C. The invention provides germplasm resources and theoretical support for cultivating new rice
varieties with early heading and good photomorphogenesis. It has broad application prospects.
Description
Rice Flowering-related Gene LHD3 and Its Application
The invention belongs to the field of plant genetic engineering. Particularly, the invention
relates to the cloning of rice LHD3(Late Heading Date 3) gene by utilizing a map-based
cloning technology and the identification of the function of the gene by utilizing a
transgenic complementary experiment. Meanwhile, the invention also relates to the
regulation and control of rice photomorphogenesis by utilizing the gene. The gene is used
for improving rice varieties to increase yield.
High yield is an eternal topic in rice breeding. Heading date is one of the most important
agronomic traits of rice. It directly affects the length of growth period and the formation
and yield of rice grains. Especially in double-cropping rice areas, the heading date of rice
is more stringent. Regulation of the flowering time of rice is one of the important means to
ensure food security in China. Therefore, the study of different flowering mutants is of
great significance to reveal the regulation mechanism of rice flowering and provide a new
way to regulate rice flowering by biotechnology.
The research on the regulation way of controlling flowering has been thoroughly studied
in the dicotyledonous model plant Arabidopsis thaliana. More than 80 genes that related
to the regulation of flowering time of Arabidopsisthalianahave been found. The regulation
of these genes is the result of the interaction of external environmental factors and
endogenous signal molecules. The interaction between these genes constitutes a fine
regulation network. As a monocotyledonous plant, the flowering regulation network of rice
is quite different from that of Arabidopsis thaliana. The differences including participating genes and regulation modes. The flowering regulation pathway of OsGI-Hdl
Hd3a in rice is similar to that of GI-CO-FT in Arabidopsis thaliana. But the difference is
that CO promotes flowering in Arabidopsis thaliana, while Hdl promotes flowering in
short-day and inhibits flowering in long-day. Under long-day conditions, Hdl inhibits
flowering by regulating Hd3a through Ghd7, Hd5/DTH8/Ghd8. There is also a flowering
regulation pathway in rice, Ghd7-Ehdl-Hd3a/RFT1, which Arabidopsis thalianadoes not
have.
Light is one of the most important external environmental factors affecting rice flowering.
Light also affects some other plant development processes, such as seed germination,
hypocotyl elongation, chlorophyll synthesis, leaf type and so on. Different photoreceptors
in plants receive different types of light signals. Phytochrome responds to red light and far
red light. Cytochrome and photoprotein receives blue light. UVR8 mainly responds to
ultraviolet light. Phytochrome is the most important photoreceptor molecule. There are
three phytochromes in rice: phytochrome A, phytochrome B and phytochrome C. In the
past 20 years, the mutants of these genes, including uniprocess, double process and triple
process, were isolated and constructed. This enhanced our understanding of the function of
rice phytochrome. At present, the research focuses on phytochrome A and phytochrome B.
Their functions in photomorphogenesis of rice have been clearly explained.
Because of the limited resources of phytochrome C mutant, only one insertion mutant has
been reported. The functional study of this gene has been neglected, which leads to a little
understanding of phytochrome C. Therefore, it is important to explore the new mutant
resources of phytochrome C for revealing the photomorphogenesis of rice and the
molecular mechanism of regulation.
The purpose of the present invention is to provide a rice flowering-related protein, an
encoding gene LHD3 thereof and an application thereof in rice breeding.
The invention provides a rice flowering-related protein, and the amino acid sequence
thereof is shown as SEQ ID NO: 2. The protein also includes amino acid sequences or
derivatives generated by adding, substituting, inserting or deleting one or more amino acids
or homologous sequences of other species in the amino acid sequence shown in SEQ ID
NO: 2.
The invention also provides a gene LHD3 encoding the protein. The nucleotide sequence
of the gene LHD3 is shown as SEQ ID NO: 1. The gene LHD3 also includes mutants,
alleles or derivatives generated by adding, substituting, inserting or deleting one or more
nucleotides in the nucleotide sequence shown in SEQ ID NO: 1.
The application of the above gene LHD3 in improving heading date and
photomorphogenesis of rice is as follows. As an improvement of the application of the gene
LHD3 of the present invention, rice cells are transformed with the gene having the
nucleotide sequence shown in SEQ ID NO: 1, and then the transformed rice cells are
cultivated into plants.
The rice late heading mutant of the invention is obtained by screening from EMS mutant
library of Japonica rice variety Oryza. Sativa L. spp. japonica. The mutant showed
delayed flowering under long-day conditions (Nanchang, Jiangxi) and short-day conditions
(Sanya, Hainan), and appeared the phenotype of pale-green leaves. By crossing the mutant
with wild-type Oryza. Sativa L. spp. japonica, observing the separation ratio of F 2
offspring to determine that the phenotype is caused by a gene. According to the invention, the rice flowering control gene LHD3 is cloned and separated by adopting a map-based cloning method. The LHD3 gene is derived from LOC_Os03g54084 gene by single base mutation. That is, nucleotide C at the 1730th position of SEQ ID NO.1 is mutated to A, which leads to the change of encoded amino acid. Bioinformatics analysis shows that
LHD3 encodes phytochrome C in rice.
Transgenic research with complementary functions is carried out through transgenic
technology. The results show that the transgenic rice which restores the phenotype of
mutant lhd3 to wild type is obtained by the invention. This proves that the LHD3 gene is
correctly cloned by the invention.
To sum up, the rice flowering gene LHD3 is isolated, cloned and identified, and the gene
function is verified by complementary experiment. The results of map-based cloning
showed that the gene encoded rice phytochrome C. The invention provides germplasm
resources and theoretical support for cultivating new rice varieties with early heading and
good photomorphogenesis. It has broad application prospects.
The invention will be further described with specific examples below. These descriptions
are not intended to further limit the contents of the present invention. Unless otherwise
specified in the following embodiments, the technical means used are conventional means
well known to those skilled in the art.
Unless otherwise specified, the materials and reagents used in the following embodiments
can be obtained from commercial sources.
Embodiment 1 Obtaining and phenotypic analysis of mutant materials
A mutant lhd3 with seriously delayed heading date was screened by EMS chemical mutated
japonica rice variety Oryza. Sativa L. spp. japonica. The mutant's traits have been
inherited stably after multiple generations of selfing. The flowering times of the mutant
were 197 days under long day conditions and 145 days under short sunshine. The flowering
times of normal Oryza . Sativa L . spp . japonica rice are 90 days and 81 days
respectively. In addition, compared with the wild type, the leaves of the mutant were lighter
in field conditions, and the chlorophyll content of the mutant was significantly lower than
that of the wild type Oryza. Sativa L. spp. japonica. All rice materials were planted in
the experimental field of Jiangxi Agricultural University in Nanchang City, Jiangxi
Province and the Nanfan Base of Jiangxi Agricultural University in Sanya City, Hainan
Province, with routine management.
The EMS chemical mutagenesis method specifically comprises the following steps. Firstly,
immersing seeds of Oryza. Sativa L. spp. japonica in ethyl methanesulfonate with the
concentration of 0.05-0.5mol/L for 30min. Then germinating the seeds and planting the
seeds in a field, and carrying out multiple generations of selfing.
Embodiment 2 Population construction and genetic analysis
In order to avoid the influence of genetic background differences on heading, the mutant
lhd3 was crossed withjaponica rice varieties Oryza. Sativa L. spp. japonica., Zhonghua
11 and Wuyunjing 7. As a result, all F1 plants showed normal heading phenotype, which
indicated that lhd3 was controlled by recessive nuclear genes. Statistics of segregation ratio
of F 2 segregation population (Table 1) showed that the segregation ratio of plants with
normal phenotype and plants with late heading phenotype was close to 3:1 after Chi-square test, which indicated that wax reduction and premature senescence phenotype of lhd3 were controlled by a pair of single recessive nuclear genes.
Table 1 Genetic analysis of rice brittle stalk mutant lhd3
F1I F2 Material combination Normal Mutant Normal Mutant X2 0 Pvalue phenotype phenotype phenotype phenotype lhd3/NPB 4 0 87 27 0.10 0.74 lhd3/ZHI1 4 0 76 22 0.34 0.56 I ----------- - lhd3/WYJ7 7 0 162 48 0.51 0.47
Embodiment 3 Fine mapping of LHD3 gene
In order to ensure the polymorphism of molecular markers, we selected indica rice variety
TN 1 to cross with mutant to construct a mapping population. To avoid the influence of
genetic differences on heading date sampling, we selected extremely late heading
individuals in F 2 population for mapping. SSR primers uniformly distributed on 12
chromosomes of rice stored in our laboratory were used to screen the polymorphism of the
mutant and TN 1. Then linkage analysis was carried out with 21 extremely late heading
individuals in F 2 oflhd3/TN 1, and the chromosome location of the target gene was
preliminarily confirmed. Genomic DNA was extracted by CTAB method. The specific
steps are as follows.
(1) Weigh 0.1g of rice leaves, grind them into powder with liquid nitrogen. Add 600pL of
CTAB solution (2% (m/v) CTAB, 100mmol/L Tris-Cl, 20mmol/L EDTA, 1.4mol/L NaCl
: PH8.0), water bath at 65C for 40min. Then add 600pL of chloroform: isoamyl alcohol
(volume ratio: 24:1) and mix gently. Centrifuge at 10000rpm for 5min, and transfer the
supernatant to a new centrifuge tube.
(2) Add 2/3 - 1 times volume of pre-cooled isopropanol (to 4C) to the supernatant obtained
after centrifugation in the above step (1), and mix them gently until DNA precipitates.
Centrifuge at 13000rpm for 8min, and pour out the supernatant.
(3) Wash the DNA precipitate obtained in the above step (2) with 200L of 70% (volume
concentration) hexanol.
(4) Dry the washed DNA and dissolve it in 100L of TE buffer or pure water.
(5) Detect the concentration of the DNA sample obtained in the above step (4) by ultraviolet
spectrophotometry. Detect the integrity of DNA by 0.7% agarose gel electrophoresis.
Complete and suitable DNA is used for PCR amplification. Incomplete DNA is re
extracted until complete DNA is obtained.
PCR reaction system adopts 10 L system: DNA template 1 L, 1OxPCR buffer 1 L,
forward and backward primers (10 mol/L) 0.5 L each, dNTPs 1 L, rTaq enzyme 0.2L,
and add H20 added to makeup 10 L. PCR amplification procedure is as follows: 94°C
pre-denaturation for 4 min, 94 °C denaturation for 30s, 55C - 60°C annealing for 30s
(temperature varies with primers), 72 °C extension for 30s and 40 cycles, finally, 72°C
extension for 10min. PCR products were electrophoresed in 4% agarose gel. After
electrophoresis, photos were taken on gel imager and gel was read. 186 pairs of SSR
primers screened above were used for gene linkage analysis. It was found that LHD3 was
initially mapped on chromosome 3. A new Indel marker was designed upstream and
downstream of the linkage marker. The target gene interval was locked between molecular
markers RM5172 and RM8203 with 96 individual plants.
In this region, a new molecular marker was designed again, and a total of 992 F 2 plants
were used to map the gene in the region of about 46kb between C6 and C5. See Table 2 for
primer sequences.
Table 2 Molecular markers used for fine positioning
Primer Forward primer (5 '-- 3') Reverse primer (5 '-- 3') name Cl GTGCTCCGTAGGCTCATCTC GACGACCCTTTCTGGAACAG C2 AGCTGATCTGCTCAAGCTGTGG CAGTCTCTCTCGGCCAATTAAG C C3 GACTTTGATTAGACAGAGGCC GCGCCAGGACTAAACTAAACA AAGC GC C4 ACATTTCGTGCTGAAAGACTGG GAAATCAGTGTACGTAGGATC AGAG C5 GTTCTCGATCCGCTTCAGCTGC CGTCGCTGATAGCGAGGTGGG ACC TAGG C6 AATCCATGTTAACTTATCCTAC GCCACATAATGGTACTTAATTA GC
According to the data of rice genome database (http://rice.plantbiology.msu.edu/), it was
found that there were 8 open reading frames (ORFs). We sequenced the whole genes
(including promoters and introns) of the eight genes of Oryza. Sativa L. spp. japonica
and lhd3, and compared them. We found that C--A single base mutation occurred in exon
1 of ORF4(LOC_Os03g54084), and the 577th amino acid changed from Ser to Thr.
The LHD3 gene has a nucleotide sequence shown in SEQ ID NO: 1, and the encoded
protein has an amino acid sequence shown in SEQ ID NO: 2
Embodiment 4 Plant transformation
The genomic DNA fragment from 5'-UTR to 3'-UTR of LHD3 gene in wild type Oryza.
Sativa L. spp. japonica was amplified, with a total length of 7638bp. The fragment was
ligated into binary vector pCAMBIA1300 by recombinant method.
The plasmid was transformed into Agrobacterium tumefaciens EHA105 by liquid nitrogen
freeze-thaw method and transformed into mutant rice. The callus induced by mature
embryo of mutant was used. After 2 weeks' culture in induction medium, the vigorous
callus was selected as the recipient of transformation. The EHA105 strain containing binary
plasmid vector (pCAMBIA1300-WSL5) was used to infect rice callus. They were co
cultured for 3 days in the dark at 25C, and then cultured in the screening medium
containing 50mg/L Hygromycin in the light (light intensity 13200LX, temperature 32°C)
for about 14 days. The pre-differentiated callus was transferred to differentiation medium
and cultured under light conditions (light intensity 13200LX, temperature 32C) for about
one month to obtain resistant transgenic plants. Transgenic seedlings were planted in the
field, and the phenotypes of the complementary seedlings were identified. Compared with
wild type and mutant at the same time, it was found that the phenotypes of transgenic plants
with delayed heading date had recovered, and there was no obvious difference between
transgenic plants and wild type.
The above is only a preferred embodiment of the present invention, and is not used to limit
the present invention. Any change or substitution that can be easily thought of by a person
familiar with the technical field within the technical scope disclosed by the present
invention should be covered within the protection scope of the present invention.
Therefore, the protection scope of the present invention should be subject to the protection
scope defined in the Claims.
Figure 1 The heading phenotype and flowering time of wild type and lhd3 mutant
Figure 2 A leaf color phenotype of wild type and lhd3 mutant.
Figure 3 A fine map of LHD3 gene
Figure 4 The phenotype and flowering time of transgenic rice in functional
complementationexperiment
Claims (4)
1. A rice flowering-related protein, characterized in that its amino acid sequence is shown
in SEQ ID NO: 2.
2. The rice flowering-related protein according to Claim 1, characterized in that the amino
acid sequence further comprises amino acid sequences or derivatives generated by adding,
substituting, inserting or deleting one or more amino acids or homologous sequences of
other species in the amino acid sequence shown in SEQ ID NO: 2.
3. A gene encoding the rice flowering-related protein according to Claim 1 or 2,
characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO: 1.
4. The gene according to Claim 3, characterized in that the nucleotide sequence further
comprises mutants, alleles or derivatives generated by adding, substituting, inserting or
deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO: 1.
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