AU2021107431A4 - Application of Galega orientalis gibberellin receptor gene GoGID1 for improving alfalfa biomass - Google Patents

Abstract

The invention relates to an application of Galega orientalis gibberellin receptor gene GoGID1 for improving alfalfa biomass, which specifically comprises the following steps: inserting the GoGID1 gene into a vector plasmid DNA; Introducing the vector inserted with GoGID1 gene into Agrobacterium tumefaciens LBA4404; Cultivation of Agrobacterium; Preparation and transformation of alfalfa explants: Identification and propagation of transgenic alfalfa plants; Evaluation of biomass-related traits and forage quality of alfalfa transgenic lines. Over-expression of gibberellin receptor gene GoGID1 in Galega orientalis in alfalfa increased the plant height of transgenic alfalfa by 0.2-0.35 times, the biomass increased by 14.9-46.3%, and the flowering time was delayed by one week compared with the control. The invention is of great significance for exploring the possibility of improving alfalfa biomass related traits, so as to improve the yield of alfalfa, an important legume pasture, and also provides possible candidate genes and theoretical basis for genetic improvement of alfalfa yield traits. 1

Description

DESCRIPTION Application of Galega orientalis gibberellin receptor gene GoGID1 for

improving alfalfa biomass

TECHNICAL FIELD

The invention relates to the field of molecular biology, in particular to an

application of Galega orientalis gibberellin receptor gene GoGID] for improving

alfalfa biomass.

BACKGROUND

Gibberellin is a kind of tetracyclic diterpene alkanes, which belongs to one of the

five major hormones in plants and plays a role in many important growth and

development processes of plants. Including seed germination, leaf extension, stem

elongation, flowering, and seed development. Gibberellin receptor is an important

link in gibberellin signaling pathway, which can combine with active GA to form

GID-GA-DELLA complex, which is recognized by ubiquitin E3 enzyme complex

(SCFGID2/SLYl), mediates DELLA protein to form polyubiquitin chain, and then is

degraded by 26S proteasome, thus triggering GA response,

Gibberellin Insensitive Dwarfl (GIDI) gene was first discovered from

gibberellin insensitive dwarf mutant in rice in 2005. The gene encodes a soluble

protein similar to hormone sensitive lipase, which is located in cytoplasm and nucleus.

Subsequently, gibberellin receptors were also found in Arabidopsis thaliana, cotton,

poplar and other species. Many studies have shown that gibberellin receptor is

involved in many plant growth and development processes, and GID mutation leads

to the classic plant dwarfing phenotype,

alfalfa (Medicago sativa) is a perennial herb of the genus alfalfa in LeguminosaE,

Because of its high yield, good palatability and good quality, it is widely planted all

over the world, and is known as the "king of pasture". The planting area in the world

DESCRIPTION exceeds 10 million hectares. The main utilization parts of alfalfa are leaves and stems,

so biological yield is an important breeding goal of alfalfa, Conventional breeding

methods have long breeding time and no obvious effect. With the development of

genomics and molecular biology technology, it has become possible to use genetic

engineering technology to improve pasture molecules. Genetically improving alfalfa

and increasing its biomass will have a far-reaching impact on forage industry.

GA receptor gene GoGID] was isolated from Galega orientalis, a perennial

legume closely related to alfalfa, Galega orientalis is an important perennial legume

forage with high yield and rich nutrition, and its feeding and utilization period is

earlier than alfalfa, Young ruminants should not suffer from bloating, and it can

promote milk secretion of dairy cows. GoGID] gene can improve biomass yield of

transgenic tobacco. With the help of genetic transformation of alfalfa, we introduced

exogenous gene GoGID] into alfalfa genome, which is of great significance for

exploring the possibility of improving the related traits of alfalfa biomass, and thus

increasing the yield of alfalfa, an important leguminous forage,

SUMMARY

To solve the problems raised in the above background art, the purpose of the

present invention is to provide the application of the gibberellin receptor gene

GoGID]of Galega orientalisto improve the biomass of alfalfa,

In order to solve the above technical problems, the technical scheme provided by

the invention is as follows: the application of Galega orientalis gibberellin receptor

gene GoGID1 to improve the alfalfa biomass, Galega orientalis gibberellin receptor

gene GoGIDlis prepared from Galega orientalis as a raw material, and the

preparation method comprises the following steps:

A, extraction of total RNA from Galega orientalis;

DESCRIPTION B, RACE clone the full length cDNA of GoGID] gene;

C, transforming the recombinant plasmid into Escherichia coli and sequencing.

Furthermore, step A of the preparation method of the gibberellin receptor gene

GoGID] of Galega orientalis adopts Trizol reagent method, which comprises the

following specific steps:

a, adding Galega orientalis tissue material into Trizol reagent, adding liquid

nitrogen, grinding quickly, and putting into homogenate;

b, sucking homogenate into a centrifuge tube, shaking violently and mixing

uniformly, standing at room temperature, and then centrifuging;

c, taking supernatant, adding chloroform, shaking violently, standing at room

temperature, and centrifuging;

d, after the solution is layered, the upper lay is taken and transferred to another

centrifugal tube, chloroform with equal volume is added, and the previous step is

repeat;

e, taking the supernatant, adding equal volume of isopropanol, mixing evenly,

standing at room temperature, and centrifuging to obtain white RNA precipitate;

f, discarding the supernatant, adding ethanol, flushing the precipitate,

centrifuging, dumping the ethanol, and leaving the precipitate;

g. repeat the previous step, air dry, and dissolve with DEPC water;

h. performing RNA electrophoresis, detecting RNA extraction quality, and

measuring RNA concentration by using a micro nucleic acid protein analyzer.

Furthermore, the preparation method step B of Galega orientalis gibberellin

receptor gene GoGIDlcomprises the following specific steps:

(1) synthesis of RACE-ready cDNA

a, prepare buffer: 5x first chain buffer, DTT and dntpmix;

DESCRIPTION b, preparing cDNA reaction solution for 5'RACE and cDNA reaction solution for

3'RACE;

c, uniformly mixing the liquid prepared in step b, centrifuging, incubating,

cooling again, and centrifuging to collect the reaction liquid;

d, adding SMARTer IIA oligo into cDNA reaction solution of 5'RACE,

centrifuging and collecting reaction solution;

e, preparing 5'RACE and 3'RACE-Ready cDNA reaction solution: uniformly

mixing the buffer solution obtained in step a, the RNase inhibitor and the reverse

transcriptase;

f, respectively adding the reaction solution obtained in step E into the 3'RACE

reaction solution obtained in step c and the 5'RACE reaction solution obtained in step

d, mixing, centrifuging and collecting the liquid;

g. incubating the prepared reaction solution;

h. heating to complete the synthesis of Ready-cDNA;

(2) design of RACE primers

Design principles include:

a, gene specific primers GSPs meet the following conditions: 23-28bp; GC

content 50%-70%; Tm value > 70°C; it is not complementary to the 3'- end universal

primer.

b, two GSP primers should be designed, the reverse primer GSP1 is used for

'RACE and the forward primer GSP2 is used for 3' race;

According to the above design principles, two primers GSP1 and GSP2 were

designed,

(3) Rapid synthesis of cDNA ends

DESCRIPTION A, preparing a PCR Mix; ed solution: adding the following reagents into a PCR

reaction system: PCR water, 10 * advantage2 PCR buffer, dNTP Mix and 50*

advantage2 polymerase Mix; Mixing, centrifuging and collecting liquid;

B, preparing PCR reaction solution for 5'RACE: 5' race-ready cdna, 10xUPM,

GSP1, and PCR mixed solution prepared in step a;

PCR reaction solution for 3'RACE was prepared: 3' race-ready cdna, 10xUPM,

GSP2, and PCR mixture prepared in step a;

C.RACE amplification:

The reaction system is: 94C for 30 seconds, 72C for 3 minutes, a total of 5

cycles; 94C for 30 seconds, 70°C for 3 minutes, 72C for 3 minutes, a total of 5

cycles; 20 cycles at 94C for 30 seconds, 68C for 30 seconds and 72C for 3 minutes;

PCR products were detected by electrophoresis on agarose gel, and the target

band was recovereD, the recovered products were ligated with T4DNA ligase on

PMD18-T vector, and transformed into Escherichia coli DH5a competent cells.

Furthermore, the primers GSP1 and GSP2 in step B of the preparation method of

Galega orientalis gibberellin receptor gene GoGIDlare shown in SEQ ID NO:1 and

SEQ ID NO:2 in the sequence table,

Furthermore, the preparation method step C of Galega orientalis gibberellin

receptor gene GoGIDlcomprises the following specific steps:

a, uniformly coating the transformed Escherichia coli solution on an LB plate

and culturing overnight in an incubator;

b, selecting white single colony in LB liquid culture medium, and culturing

overnight;

c, carrying out PCR detection on the bacterial liquid, selecting positive clones

and sequencing.

DESCRIPTION Furthermore, the method for transforming alfalfa by GoGID1 gene comprises the

following steps:

(1) inserting GoGID1 gene into vector plasmid DNA:

The vector is PBI 121 and comprises the following specific steps:

The upstream primer P1 with XbaI restriction site and the downstream primer P2

with BamHI restriction site at the 5' end are designed as shown in SEQ ID NO:5 and

SEQ ID NO:6 in the sequence table,

Pl: 5'-GCTCTAGAATGGTGATCGAGAACGACATTGAG-3';

P2: 5'-CGGGATCCTCAGTGATGGCCACGCCTAAGTGATG-3';

Among them, the underlined part is the enzyme digestion site;

The full-length reading frame of GoGID1 was amplified from Galega orientalis

cDNA template with this primer. PBI vector and full-length fragment of target gene

were digested by XbaI and BamHI, and then the products were recovereD, after T4

DNA ligase connection, PB1121 vector with GoGID1 gene inserted was obtained,

which contained CaMV35s promoter, GoGID gene and a kanamycin resistance

screening marker.

(2) introducing the vector inserted with GoGID1 gene into Agrobacterium

tumefaciens LBA4404, which comprises the following specific steps:

A, adding plasmid DNA into Agrobacterium tumefaciens competent cell

LBA4404, mixing uniformly, and taking an ice bath;

B, quickly freezing in liquid nitrogen, and immediately placing in a water bath

for incubation;

C, adding YEB liquid culture medium and culturing;

DESCRIPTION (3) Cultivation of Agrobacterium; The Agrobacterium with GoGID] gene

introduced is plated on a solid medium containing kanamycin and streptomycin,

placed in an incubator and cultured;

Selecting single colony on the plate, inoculating in YEB liquid medium

containing kanamycin and streptomycin, and culturing; Take a small amount of

bacterial liquid, dilute it into YEB liquid medium containing kanamycin and

streptomycin, and culture it until logarithmic growth period;

Collecting thalli in a centrifuge tube, centrifugally enriching thalli, discarding

supernatant, and resuspending thalli in a modified SH liquid culture medium until the

OD600 value of the bacterial liquid is 0.6-0.8 for later use;

(4) Preparation and transformation of alfalfa explants; Taking alfalfa as material,

growing in artificial climate room, taking dark green healthy leaves and cutting them

into explants;

The explant was put in Agrobacterium suspension, then put on sterile filter paper

to remove excess liquid;

Growing on a co-culture medium; Take out that explant and sterilizing; And then

transfer to a screening culture medium;

Embryogenic callus will be induced by transferring callus into induction medium;

Continue subculture, embryogenic callus differentiates into buds and forms

seedlings;

Transferring seedlings to rooting culture medium, continuing to culture until

rooting, hardening seedlings, transferring into soil, and culturing into plants in

greenhouse;

DESCRIPTION (5) Identification and propagation of alfalfa transgenic plants: DNA was

extracted from the genetically transformed plants, and was detected by npt II ii gene

primers P3 and P4 and GUS gene primers P5 and P6;

RNA transcription level was detected by primers P7 and P8, among which P3-P6

were shown as SEQ ID NO:7-NO:10 in the sequence table,

Selecte transgenic lines with high expression level to grow in a large flowerpot,

and propagate by cuttage after that plants grow up;

(6) Evaluation of biomass-related traits and forage quality of alfalfa transgenic

plants: Agrobacterium tumefaciens was transformed with plasmid PBI121 to obtain

recombinant Agrobacterium tumefaciens, and Arabidopsis thaliana was transformed

with recombinant Agrobacterium tumefaciens to obtain control alfalfa plants with

empty vectors;

The transgenic lines and control lines were propagated by cuttage, and the

cuttage plants were grown in the greenhouse, During the period, they were carefully

managed, watered regularly, and the diseases and insect pests were controlled in timE,

Cut 2-3 times during the period to make the plants in the same statE, At the initial

flowering stage of alfalfa, 10 cuttings were randomly selected from each material

(transgenic material and control plant), and the phenotypes were observeD, The

indexes such as plant height, fresh weight, dry weight, leaf length, leaf width, stem

node number and flowering period were measured, and the forage quality indexes

such as CP, ADF and NDF were also measured,

The method has the beneficial effects that: after the gibberellin receptor gene

GoGID]of the Galega orientalis is overexpressed in the alfalfa, the plant height of

the transgenic alfalfa is increased by 0.2-0.35 times, the leaf length and width are

obviously higher than those of the control, the biomass is increased by 14.9-46.3%,

DESCRIPTION and the flowering time is one week later than that of the control. Moreover, the

quality index of transgenic alfalfa was not significantly affecteD, The results showed

that GoGID1, as a gibberellin receptor gene, regulated many biomass-related traits of

alfalfa, and delayed the flowering period of transgenic alfalfa, thus increasing the

biomass of transgenic alfalfa, The method has important significance for exploring the

possibility of improving alfalfa biomass related traits, thereby improving the yield of

alfalfa, an important leguminous pasture, and providing possible candidate genes and

theoretical basis for genetic improvement of alfalfa yield traits.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic diagram of plant expression vector construction in an

embodiment of the present invention.

DESCRIPTION OF THE INVENTION

The following is a description of the present invention with specific examples,

which are not intended to limit the present invention.

Example 1

Preparation of gibberellin receptor gene GoGID] from Galega orientalis;

According to the data of differential hybridization library of Galega orientalis,

RACE amplification primers GSP1 and GSP2 were designed, as shown in SEQ ID

NO:1 and SEQ ID NO:2 in the sequence table, the sequences were cloned by 5'RACE

and 3'RACE with smartertm race cdna amplification kit (clontech, USA), and finally

the full-length sequence of the gene was obtaineD, Specific methods are as follows:

1. extraction of total RNA from Galega orientalis:

Trizol reagent method was used to extract total RNA from Galega orientalis, and

the specific steps were as follows:

DESCRIPTION (1) adding 0.3g of Galega orientalis tissue material into 3000d1Trizol reagent,

adding liquid nitrogen, grinding quickly, and placing in sterile environment until it

becomes homogenate;

(2) Suck lmL homogenate into a 1.5mL centrifuge tube with a pipette, shake it

violently and mix it evenly, then place it at room temperature for 5min, and then

centrifuge it for 10min;at 13000rpm at 4C;

(3) taking supernatant, adding 200uL chloroform, shaking violently, standing at

room temperature for 2-3min, and then decentering at 13000rpm at 4C for 15min;

(4) The solution is divided into three layers from bottom to top: organic phase,

protein and colorless water phase; Transfer the upper layer to another branch tube,

add equal volume of chloroform, and repeat the previous step;

(5) taking the supernatant, adding equal volume of isopropanol, mixing evenly,

standing at room temperature for 10min, and then centrifuging at 13000rpm at 4C for

min to obtain white RNA precipitate;

(6) discard the supernatant, add 1000L of 75% ethanol prepared from DEPC

water, flush the precipitate, centrifuge at 13000rpm at 4C for 10min, pour out the

ethanol and leave the precipitate;

(7) repeat the previous step, dry in sterile environment, and add 20-50 sterile

DEPC water for dissolution;

(8) RNA electrophoresis was carried out to detect the quality of RNA extraction,

and RNA concentration was measured by micro nucleic acid protein analyzer.

2. full length of cDNA of RACE clone gene

(1) synthesis of RACE-ready cDNA

DESCRIPTION a, prepare buffer: 2.0 1 of 5x first chain buffer, 1.0t1 of DTT and 1.0t1 of

dntpmix; Among them, the concentration of DTT is 20mM, and the concentration of

dNTP Mix is 10mM;

b, preparing cDNA reaction solution for 5'RACE: 2.75tl RNA, 1.0 1 5'-CDs

primer A; A cDNA reaction solution for 3'RACE was prepared: 3.75tl RNA, 1.0 1

3'-CDs primer A;

c, uniformly mixing the liquid prepared in step b, centrifuging briefly, incubating

at 72 °C for 3 minutes, cooling at 42 °C for 2 minutes, and centrifuging briefly to

collect the reaction liquid;

d, adding 1 1 of SMARTer IIA oligo into the cDNA reaction solution of 5'RACE,

and centrifuging for a short time to collect the reaction solution;

e, preparing 5'RACE and 3'RACE-Ready cDNA reaction solution: 4.0jl of

buffer solution obtained in step A, 0.25d1of RNase inhibitor and 1.0 l of reverse

transcriptase, and mixing the above reagents evenly; Among them, the concentration

of RNase inhibitor is 40 U/ L; The concentration of reverse transcriptase was 100

U/p L;

f, adding 5.25d1 of the reaction solution obtained in step E to the 3'RACE

reaction solution obtained in step C and the 5'RACE reaction solution obtained in step

D, respectively, gently mixing, and centrifuging for a short time to collect the liquid;

g. incubating the prepared reaction solution at 42 °C for 90 minutes;

h. heating at 70°C for 10 minutes to complete the synthesis of Ready-cDNA;

(2) design of RACE primers

Design principles include:

DESCRIPTION a, gene specific primers GSPs meet the following conditions: 23-28bp; GC

content 50%-70%; Tm value > 70°C; It is not complementary to the 3'- end universal

primer.

b, two GSP primers should be designed, the reverse primer GSP1 is used for

'RACE and the forward primer GSP2 is used for 3' race;

According to the above design principles, two primers, GSP1 and GSP2, were

designed as shown in SEQ ID NO:1 and SEQ IDNO:2 in the sequence table,

(3) Rapid synthesis of cDNA ends

a, preparing PCR mixed solution: adding the following reagents into every 50[1

PCR reaction system: 34.5[l PCR water, 5.01 lOxAdvantage 2PCR buffer, 1.0 l

dNTP Mix, 1.0jl 50xAdvantage 2 polymerase mix; Gently mix the liquid, do not

produce bubbles, and centrifuge for a short time to collect the liquid; In which the

concentration of dNTP Mix is 10mM;

b, preparing PCR reaction solution for 5'RACE: 2.5 1 2.5tl 5'RACE-ready

cDNA, 5.0tl1 OxUPM, 1.0 1GSP1; 41.5pl of the PCR mixture prepared in step A;

PCR reaction solution for 3'RACE was prepared: 2.5 1 3' race-ready cdna,

5.0pl 1OxUPM, 1.0[t 1GSP2; 41.5tlof the PCR mixture prepared in step A;

c.RACE amplification:

The reaction system is: 94C for 30 seconds, 72C for 3 minutes, a total of 5

cycles; 94C for 30 seconds, 70°C for 3 minutes, 72C for 3 minutes, a total of 5

cycles; 20 cycles at 94C for 30 seconds, 68C for 30 seconds and 72C for 3 minutes;

The PCR products were detected by electrophoresis on agarose gel with a weight

percentage of 1.0%, and the target band was recovereD, The recovered products were

ligated with T4DNA ligase on PMD18-T vector and transformed into E,coli DH5a

competent cells.

DESCRIPTION Monoclonal selection, inoculation in LB liquid medium for overnight culture,

PCR detection of bacterial liquid, and sending positive clones to Invitrogen for

sequencing. Sequence splicing showed that the gene had the nucleotide sequence

shown in SEQ ID NO:3 in the sequence table, and the encoded amino acid sequence

was shown in SEQ ID NO:4 in the sequence table,

Example 2 Agrobacterium-mediated genetic transformation of GoGID1 gene

into alfalfa plants

1. Materials and reagents

1.1 plant materials

The alfalfa varieties tested were alfalfa (Medicago sativa L L.cv.Zhongmu No.1,

zhongmu No.1).

1.2 Agrobacterium strains and plasmid vectors

The Agrobacterium strain used is Agrobacterium tumefaciens LBA4404

Agrobacterium medium:

table 1

Reagent Content(1L)

MgS047h20 1 g/L

Peptone 10 g/L

Yeast extract 1 g/L

Sucrose 5 g/L

Agar (solid medium) 15 g/L

pH 7.0

Sterilizing with high pressure steam at 121°C for 20min; Vector: PBI121.

DESCRIPTION 2. Experimental methods

2.1 inserting the GoGID1 gene into the plasmid DNA of PB1121 vector, the

specific steps are as follows: designing an upstream primer containing XbaI

restriction site and a downstream primer containing BamHI restriction site at the 5'

end, as shown in SEQ ID NO:5 and SEQ IDNO:6 in the sequence table;

(Pl: 5'-GCTCTAGAATGGTGATCGAGAACGACATTGAG-3',

P2: 5'-CGGGATCCTCAGTGATGGCCACGCCTAAGTGATG-3',

Among them, the underlined part is the enzyme digestion site);

The primer is used to amplify the full-length reading frame of GoGID1 from

Galega orientalis cDNA templatE, After the PBI vector and the full-length fragment

of the target gene are digested by XbaI and BamHI, the products are recovered, and

then connected by T4DNA ligase, the PB1121 vector with GoGID1 gene inserted is

obtained, which contains CaMV35s promoter, GoGID1 gene and a kanamycin

resistance screening marker. The transgenic plants obtained during genetic

transformation can be preliminarily identified by kanamycin screening. The structure

of plasmid vector containing the target fragment is shown in Figure 1.

2.2 introducing PB1121 vector with GoGID1 gene into Agrobacterium

tumefaciens LBA4404, the specific steps are as follows:

A, add about 1 g plasmid DNA into 100d1Agrobacterium tumefaciens

competent cell LBA4404, mix gently, and ice bath for 30min;

B, quick-freezing in liquid nitrogen for 1min, and immediately incubating in

37°C water bath for 5 min;

C. adding 800d1YEB liquid culture medium, and culturing at 28C and

28°C 150rpm for 4-6h;

DESCRIPTION D, coating the thallus on a YEB selection plate containing 50mg/L kanamycin

and 100mg/L streptomycin, and culturing the thallus upside down at 28 °C for two

days.

E, select single colony, inoculate it in YEB liquid medium (containing 50mg/L

kanamycin and 100mg/L streptomycin), and shake culture at 28C overnight.

The Agrobacterium plasmid introduced with GoGID1 gene was extracted and

sequenceD, the results showed that the nucleotide sequence of the introduced gene

was consistent with the sequence shown in SEQ ID NO:3 in the sequence table,

indicating that the expression vector containing the target gene GoGID1 was

successfully constructeD,

2.3 Cultivation of Agrobacterium tumefaciens

The Agrobacterium with GoGOD1 gene was crossed on the solid culture

medium containing 50mg/L kanamycin and 100mg/L streptomycin, and cultured in an

incubator at 28C. Two days later, the single colony was picked from the plate and

inoculated in 20ml YEB liquid medium containing 50mg/L kanamycin and 100mg/L

streptomycin, and cultured at 180rpm at 28C. Scratch the plate with the cultured

bacterial solution, culture at 28C, and store the plate at 4C after growing single

colony.

Selecting single colony on the plate, inoculating it into 20ml YEB liquid medium

containing 50mg/L kanamycin and 100mg/L streptomycin, and culturing on a

constant temperature shaker at 28C and 180rpm. Two days later, a small amount of

bacterial liquid was taken, diluted into YEB liquid medium containing 50mg/L

kanamycin and 100mg/L streptomycin at a ratio of 1:50-1:100, and cultured at 28C

for 6-12h to logarithmic growth perioD, Collect the bacteria in a centrifuge tube,

DESCRIPTION centrifuge at 4000rpm for 10min to enrich the bacteria, discard the supernatant, and

then use about 20ml of modified SH liquid culture medium without antibiotic to

resuspend the bacteria until the OD600 value of the bacteria liquid is 0.6-0.8.

2.4. preparation of explants:

alfalfa plants were used as materials, and grown in artificial climate room. After

3-4 weeks, leaves (dark green healthy leaves) were cut into explants under aseptic

conditions.

2.5. transformation process

Infect explants in A600 0.6-0.8 Agrobacterium suspension (sterilized YEP) for

-30 minutes, then put them on sterile filter paper to remove excess liquid; Growing

on co-culture medium for 7-8 days; Take out the explant, put it in sterilized water, and

turn it upside down for 10-20 times to remove bacteriA, Repeat the above process 2-3

times. And then transfer to a screening medium; After 2-3 weeks, the induced callus

was transferred to the induction medium; After 3 -4 weeks, embryogenic callus will

be induced, and subculture will continue, and embryogenic callus will differentiate

into buds and form seedlings; The seedlings were transferred to rooting medium to

continue to be cultivated until they took root. After hardening, they were transferred

to soil and cultivated into plants in greenhouse,

2.6 Identification of Transgenic Positive Plants

Genomic DNA was extracted from some leaves of the above-mentioned

transformed plants, and RNA transcription level was detected with npt II ii gene

primers P3 and P4, GUS gene primers P5 and P6 and primers P7 and P8. Among

them, P3 - P8 are shown as SEQ ID NO: 7 - NO: 12 in the sequence table;

Transgenic lines L9, L38 and L39 with high expression were selected and grown

in vats. After growing up, the propagation plants were obtained by cuttage,

DESCRIPTION Example 3 evaluation of biomass related traits and forage quality of GoGID]

transgenic alfalfa

1. Materials and reagents

1.1 plant materials

The tested materials were transgenic positive lines of alfalfa transformed into

GoGID1, and alfalfa transformed into empty vector as control.

2. Experimental methods

2.1 construction of control lines:

Agrobacterium tumefaciens was transformed with PBI121 vector to obtain

recombinant Agrobacterium tumefaciens, and then transformed with recombinant

Agrobacterium tumefaciens to obtain the control strain of empty vector. The method

was as follows: step 2.2 - 2.6, and the control strain of empty vector (CK) was

obtained as comparative example 1.

Transgenic lines L9, L38, L39 and control lines (CK) were transplanted into

large flowerpots to grow. After the plants grew up, they were propagated by cuttage,

2.2 Evaluation of the traits of transgenic materials

cuttage plants grow in greenhouse, during which they are carefully managed,

watered regularly, and pests and diseases are controlled in timE, Cut 2-3 times during

the period to make the plants in the same statE, At the initial flowering stage of alfalfa,

cuttings were randomly selected from each material (transgenic material and

control plant), and the phenotypes were observeD, The indexes such as plant height,

fresh weight, dry weight, leaf length, leaf width, stem node number and flowering

period were measured,

Specifically:

DESCRIPTION Plant height: measure the absolute height of the plant from the root and neck to

the top of the main stem with a tape measure as the plant height in centimeters,

calculate the arithmetic mean as the measured value, and keep 2 decimal places for

the data,

Branch number: measure the number offirst-order branches and second-order

branches at the initial flowering stage, The stems and branches directly extracted from

the root and neck of alfalfa are thefirst-class branches, and the branches extracted

from the axils of thefirst-class branches are the second-class branches. Ten individual

plants were measured for each material, and the arithmetic mean was calculated as the

final measured value,

Leaf length: 10 main stems and leaves are selected from each material. The

method is to measure the length from the base of the leaf to the tip of the leaf with a

ruler, in cm, and calculate the arithmetic mean as the final measured value, and keep 2

decimal places for the data,

Leaf width: 10 main stems and leaves were randomly selected from each plant,

and the width at the widest part of the leaves was measured with a ruler, in cm, with 2

decimal places reserved,

Intemode number: record the number of all stem nodes on the main stem of each

plant, and calculate the arithmetic mean as the final measured value,

Dry matter content: After measuring all the above indexes, cut all the

aboveground parts of a single plant (pay attention to the same stubble height),

measure the fresh weight of a single plant, deactivate the enzyme at 105°C, then dry

the plant samples in an oven at 65C to a constant weight, and weigh them as dry

weight. Calculate the arithmetic mean as the final measured value, in g, and keep 2

decimal places for data,

DESCRIPTION Flowering period: observe carefully and record in detail the date of the first

flowering period of each material and plant.

See table 2 for statistical results.

2.3 quality evaluation of genetically modified materials

The dried samples were crushed and sieved with 40 mesh sieve for determination

of nutritional components.

The content of Crude protein ( CP ) was determined according to Kjeldahl

method, and the content of Neutral detergent fiber(NDF) and Acid detergent fiber,

(ADF) was determined according to normal washing fiber methoD, Each strain has

three biological repetitions, and each biological repetition has three technical

repetitions. Calculate the arithmetic mean as the final measured value, and keep two

decimal places in the data,

See table 3 for statistical results.

table 2 Biomass related traits of transgenic lines and comparative examples

Strain Plant Dry Leaf Leaf Branch Number Flowerin height(cm) weight(g) length(mm) width(mm) number internoditime(day)

comparative 70.75+5.74 6.37+1.22 22.71+1.63 9.74+1.19 7.25+0.96 14-16 25+2 example 1 L9 84.50+2.52** 7.99+0.34** 25.54+1.73** 11.89+0.87** 9.75+0.96** 14-16 29+2

L38 95.50+4.93** 9.32+0.78** 23.50+1.70** 10.50+0.78** 9.75+1.71** 13-15 28+2 L39 87.00+6.68** 8.30+0.96** 24.67+1.23** 11.22+0.85** 9.25+1.26** 13-17 29+2

Note:* * indicates that there is a significant difference between transgenic

alfalfa and alfalfa transformed into empty vector.

table 3 forage quality indexes of transgenic lines and comparative examples

Strain CP (%/DW) NDF(%/DW) ADF(%/DW)

DESCRIPTION comparative 16.67±1.13 42.72±0.97 31.72±0.66 example 1 L9 16.06±0.39 42.13±0.68 30.87±0.54 L38 16.93±0.87 42.53±0.61 31.38±0.46 L39 16.75±0.79 44.32±1.90 33.30±1.08

Compared with the control alfalfa, the height of transgenic alfalfa increased by

0.2-0.35 times, the leaf length and width were significantly higher than the control

alfalfa, the biomass increased by 14.9-46.3%, and the flowering time of transgenic

alfalfa was delayed by one week. The results showed that GoGID1, as a gibberellin

receptor gene, regulated many biomass-related traits of alfalfa, delayed the flowering

period of transgenic alfalfa, and then increased the biomass of transgenic alfalfa, and

the quality of alfalfa was not affected by transgenic.

The above is only a preferred embodiment of the present invention, but the

protection scope of the present invention is not limited to this. Any person familiar

with the technical field who makes equivalent substitution or change according to the

technical scheme and inventive concept of the present invention within the technical

scope disclosed by the present invention should be covered within the protection

scope of the present invention.

SEQUENCE LISTING Aug 2021

< 110 > Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences

< 120 > Method for preparing gibberellin receptor gene GoGID of Galega orientalis and method for transforming alfalfa <130> None 2021107431

<160> 12

<170> PatentIn version 3.5

<210> 1 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> Reverse primer GSP1

<400> 1 aggttgggct atgctcaaag ttcc 24

<210> 2 <211> 27 <212> DNA <213> Artificial Sequence

<220> <223> Forward primerGSP2

<400> 2 ggacagtgct acttcgtgtt ctatgcg 27

<210> 3 <211> 1732 <212> DNA <213> Galega orientalis

<400> 3 acatggggac tgttactttt taagtttatt tttgtttgga ttttgttttt tcaaatcact 60

cttgttctat agatattcat attttagaga gagagagaaa gaaagaacat agagacatga 120

ctggaagtaa tgaagtcaac ctcagtgaat ctaagagtgt tgttcctctc aatacttggg 180

tgcttatctc caatttcaag ctagcttaca atctacttag acgtgctgat ggaacattca 240

acagagagct agctgagttt ctcgaccgca aagtcccagc caatacaatt cctgttgatg 300 2021107431

gagtattctc ttttgatcat gtcgatcgaa ataccggact atttagtagg gtttatcaac 360

ctgcttctga aaatgtaact acttggggca ttatagagct agaaaagccc ttgagcacaa 420

cagagattgt tcctgtcata attttcttcc atggtggaag cttctctcat tcctcagcta 480

atagtgctat ttacgacact ttctgccgtc gcctcgtgag catgtgtaag gctgttgttg 540

tttctgtcaa ctaccggaga tcacccgagc atcgttatcc gtgtgcttat gaagatggtt 600

ggaatgctct tcagtgggtt aaatcaagga catggcttca aagtggtaaa gattctaagg 660

tttatgttta catggctggt gatagttctg gtggaaacat tgcccatcat gtcgcggtga 720

gagctgccga ggaagatgtt gaggtactag gtaatattct tctccatccg cttttcggcg 780

gcgagaggcg aaccgaatcg gagaagaaat tggatggaaa atattttgtt aggttgcaag 840

atcgcgattg gtattggaga gcttttctac ctgaaggaga agatagagat catcctgctt 900

gtaatccatt tggtcctaaa ggtaaaagcc ttgcaggact caagtttgct aaaagccttg 960

tttgtgtggc tggtttggat cttcttcaag attggcaatt ggaatatgtg gaaggtctca 1020

agagttttga ccaagatgtc aaacttcttt acctaaagga agctacaatt ggtttctact 1080

tcttgcctaa taatgatcat ttctattgcc tctttaatga gataaacacc tttgtgcacc 1140

ctaactgtta atatactact actactacta cttctacaac ttaacctttg cctacacaag 1200

gccatacatg actaaaagcc tgactttttc actagtgtta tctttctttt ttatcctttt 1260

tttttgtttt ttttatctcc ctactatata gtggtggtgt gtagttataa tattattatg 1320

tagcattact cactgtttta acatctatgg tggtaaatcc gagctttatg taatcggtgg 1380

acagtgctac ttcgtgttct atgcgtctat cttttgaagg gtagcgccag tttccggcgt 1440

atgactacga ggtactggag ctcagtacat taacggttac caacttgttc gacctcgaaa 1500

ggagaggtga gatacaagat agggcttggc tgatcactct gattgagtgt ttttatgttg 1560

tataactatt atatagagtg ggaactttga gcatagccca acctgttatg aacttgtatg 1620 2021107431

ttatatatat agtaatataa atatgtcttg tttgtttgct tttgaaatta tatttttgca 1680

aaactaatat tgtttgtagt tcttaaaaaa aaaaaaaaaa aaaaaaaaaa aa 1732

<210> 4 <211> 344 <212> PRT <213> Galega orientalis

<400> 4

Met Thr Gly Ser Asn Glu Val Asn Leu Ser Glu Ser Lys Ser Val Val 1 5 10 15

Pro Leu Asn Thr Trp Val Leu Ile Ser Asn Phe Lys Leu Ala Tyr Asn 20 25 30

Leu Leu Arg Arg Ala Asp Gly Thr Phe Asn Arg Glu Leu Ala Glu Phe 35 40 45

Leu Asp Arg Lys Val Pro Ala Asn Thr Ile Pro Val Asp Gly Val Phe 50 55 60

Ser Phe Asp His Val Asp Arg Asn Thr Gly Leu Phe Ser Arg Val Tyr 65 70 75 80

Gln Pro Ala Ser Glu Asn Val Thr Thr Trp Gly Ile Ile Glu Leu Glu 85 90 95

Lys Pro Leu Ser Thr Thr Glu Ile Val Pro Val Ile Ile Phe Phe His 100 105 110

Gly Gly Ser Phe Ser His Ser Ser Ala Asn Ser Ala Ile Tyr Asp Thr 115 120 125 2021107431

Phe Cys Arg Arg Leu Val Ser Met Cys Lys Ala Val Val Val Ser Val 130 135 140

Asn Tyr Arg Arg Ser Pro Glu His Arg Tyr Pro Cys Ala Tyr Glu Asp 145 150 155 160

Gly Trp Asn Ala Leu Gln Trp Val Lys Ser Arg Thr Trp Leu Gln Ser 165 170 175

Gly Lys Asp Ser Lys Val Tyr Val Tyr Met Ala Gly Asp Ser Ser Gly 180 185 190

Gly Asn Ile Ala His His Val Ala Val Arg Ala Ala Glu Glu Asp Val 195 200 205

Glu Val Leu Gly Asn Ile Leu Leu His Pro Leu Phe Gly Gly Glu Arg 210 215 220

Arg Thr Glu Ser Glu Lys Lys Leu Asp Gly Lys Tyr Phe Val Arg Leu 225 230 235 240

Gln Asp Arg Asp Trp Tyr Trp Arg Ala Phe Leu Pro Glu Gly Glu Asp 245 250 255

Arg Asp His Pro Ala Cys Asn Pro Phe Gly Pro Lys Gly Lys Ser Leu 260 265 270

Ala Gly Leu Lys Phe Ala Lys Ser Leu Val Cys Val Ala Gly Leu Asp 275 280 285

Leu Leu Gln Asp Trp Gln Leu Glu Tyr Val Glu Gly Leu Lys Ser Phe 290 295 300 2021107431

Asp Gln Asp Val Lys Leu Leu Tyr Leu Lys Glu Ala Thr Ile Gly Phe 305 310 315 320

Tyr Phe Leu Pro Asn Asn Asp His Phe Tyr Cys Leu Phe Asn Glu Ile 325 330 335

Asn Thr Phe Val His Pro Asn Cys 340

<210> 5 <211> 32 <212> DNA <213> Artificial Sequence

<220> <223>The 5' end contains the upstream primer ofXbaI restriction site

<400> 5 gctctagaat ggtgatcgag aacgacattg ag 32

<210> 6 <211> 34 <212> DNA <213> Artificial Sequence

<220> <223> The 5' end contains the downstream primer of BamHI restriction site

<400> 6 cgggatcctc agtgatggcc acgcctaagt gatg 34

<210> 7 <211> 28 <212> DNA <213> Artificial Sequence

<220> <223> nptⅡGene primer 2021107431

<400> 7 atgattgaac aagatggatt gcacgcag 28

<210> 8 <211> 25 <212> DNA <213> Artificial Sequence

<220> <223> nptⅡGene primer

<400> 8 tcagaagaac tcgtcaagaa ggcga 25

<210> 9 <211> 27 <212> DNA <213> Artificial Sequence

<220> <223> GUSGene primer

<400> 9 atgttacgtc ctgtagaaac cccaacc 27

<210> 10 <211> 22 <212> DNA <213> Artificial Sequence

<220>

<223> GUSGene primer Aug 2021

<400> 10 tcattgtttg cctccctgct gc 22

<210> 11 <211> 21 <212> DNA 2021107431

<213> Artificial Sequence

<220> <223> Primer

<400> 11 gcaaagtccc agccaataca a 21

<210> 12 <211> 23 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 12 cggcagaaag tgtcgtaaat agc 23

Claims (5)

1. Claims 1. Application of Galega orientalis gibberellin receptor gene GoGID1 to

   improve alfalfa biomass is characterized in that Galega orientalis gibberellin receptor

   gene GoGIDlis prepared from Galega orientalis as raw material, and the preparation

   method comprises the following steps:

   A, extraction of total RNA from Galega orientalis;

   B, RACE clone the full length cDNA of GoGID1 gene;

   C, transforming the recombinant plasmid into Escherichia coli and sequencing.
2. 2. The application of Galega orientalis gibberellin receptor gene GoGIDI to

   improve alfalfa biomass according to claim 1 is characterized in that step A of the

   preparation method of Galega orientalis gibberellin receptor gene GoGID1adopts

   Trizol reagent method and comprises the following specific steps:

   a, adding Galega orientalis tissue material into Trizol reagent, adding liquid

   nitrogen, grinding quickly, and putting into homogenate;

   b, sucking homogenate into a centrifuge tube, shaking violently and mixing

   uniformly, standing at room temperature, and then centrifuging;

   c, taking supernatant, adding chloroform, shaking violently, standing at room

   temperature, and centrifuging;

   d, after the solution is layered, the upper lay is taken and transferred to another

   centrifugal tube, chloroform with equal volume is added, and the previous step is

   repeat;

   e, taking the supernatant, adding equal volume of isopropanol, mixing evenly,

   standing at room temperature, and centrifuging to obtain white RNA precipitate;

   f, discarding the supernatant, adding ethanol, flushing the precipitate,

   centrifuging, dumping the ethanol, and leaving the precipitate;

   g. repeat the previous step, air dry, and dissolve with DEPC water;

   Claims h. performing RNA electrophoresis, detecting RNA extraction quality, and

   measuring RNA concentration by using a micro nucleic acid protein analyzer.
3. 3. The application of Galega orientalis gibberellin receptor gene GoGIDI to

   improve alfalfa biomass according to claim 1, characterized in that the preparation

   method step B of Galega orientalis gibberellin receptor gene GoGIDlcomprises the

   following specific steps:

   (1) synthesis of RACE-ready cDNA

   a, prepare buffer: 5x first chain buffer, DTT and dntpmix;

   b, preparing cDNA reaction solution for 5'RACE and cDNA reaction solution for

   3'RACE;

   c, uniformly mixing the liquid prepared in step b, centrifuging, incubating,

   cooling again, and centrifuging to collect the reaction liquid;

   d, adding SMARTer IIA oligo into cDNA reaction solution of 5'RACE,

   centrifuging and collecting reaction solution;

   e, preparing 5'RACE and 3'RACE-Ready cDNA reaction solution: uniformly

   mixing the buffer solution obtained in step a, the RNase inhibitor and the reverse

   transcriptase;

   f, respectively adding the reaction solution obtained in step E into the 3'RACE

   reaction solution obtained in step c and the 5'RACE reaction solution obtained in step

   d, mixing, centrifuging and collecting the liquid;

   g. incubating the prepared reaction solution;

   h. heating to complete the synthesis of Ready-cDNA;

   (2) design of RACE primers

   Design principles include:

   Claims a, gene specific primers GSPs meet the following conditions: 23-28bp; GC

   content 50%-70%; Tm value > 70°C; it is not complementary to the 3'- end universal

   primer.

   b, two GSP primers should be designed, the reverse primer GSP1 is used for

   'RACE and the forward primer GSP2 is used for 3' race;

   According to the above design principles, two primers GSP1 and GSP2 were

   designed,

   (3) Rapid synthesis of cDNA ends

   A, preparing a PCR Mix; ed solution: adding the following reagents into a PCR

   reaction system: PCR water, 10 * advantage2 PCR buffer, dNTP Mix and 50*

   advantage2 polymerase Mix; Mixing, centrifuging and collecting liquid;

   B, preparing PCR reaction solution for 5'RACE: 5' race-ready cdna, 10xUPM,

   GSP1, and PCR mixed solution prepared in step a;

   PCR reaction solution for 3'RACE was prepared: 3' race-ready cdna, 10xUPM,

   GSP2, and PCR mixture prepared in step a;

   C.RACE amplification:

   The reaction system is: 94C for 30 seconds, 72C for 3 minutes, a total of 5

   cycles; 94C for 30 seconds, 70°C for 3 minutes, 72C for 3 minutes, a total of 5

   cycles; 20 cycles at 94C for 30 seconds, 68C for 30 seconds and 72C for 3 minutes;

   PCR products were detected by electrophoresis on agarose gel, and the target

   band was recovereD, the recovered products were ligated with T4DNA ligase on

   PMD18-T vector, and transformed into Escherichia coli DH5a competent cells.
4. 4. The application of Galega orientalis gibberellin receptor gene GoGIDI to

   improve alfalfa biomass according to claim 1, characterized in that the primers GSP1

   and GSP2 in step B of the preparation method of Galega orientalis gibberellin

   Claims receptor gene GoGIDlare shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence

   table,
5. 5. The application of Galega orientalis gibberellin receptor gene GoGIDI to

   improve alfalfa biomass according to claim 1, characterized in that the preparation

   method step C of Galega orientalis gibberellin receptor gene GoGIDlcomprises the

   following specific steps:

   a, uniformly coating the transformed Escherichia coli solution on an LB plate

   and culturing overnight in an incubator;

   b, selecting white single colony in LB liquid culture medium, and culturing

   overnight;

   c, carrying out PCR detection on the bacterial liquid, selecting positive clones

   and sequencing.

   Figure 1 DRAWINGS