CN109406730B - Screening method for mixed library gene editing T2 substitute tobacco plants - Google Patents
Screening method for mixed library gene editing T2 substitute tobacco plants Download PDFInfo
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Abstract
The invention discloses a screening method of mixed library gene editing T2 substitute tobacco plants, which comprises the following steps: (1) collecting sample information by using a palm computer and wirelessly connecting the sample information with a server terminal; (2) managing tobacco leaf samples according to the result of wireless connection between the palm computer and the server terminal, and completing operations of screening tobacco plants needing tobacco leaf sample collection, signing, processing, storage, sample sending, detection, remaining sample storage and remaining sample treatment in real time; and feeding the detection result back to tobacco plant growers, thereby completing the screening of mixed library gene editing T2 substitute tobacco plants. The method can achieve efficient and accurate screening of similar massive mixed library gene editing T2 generation tobacco strains.
Description
Technical Field
The invention relates to the field of computer software, in particular to a method for screening massive tobacco plants for efficient and accurate mixed database gene editing T2 generation in gene editing breeding.
Background
Gene editing breeding techniques have been widely used in genome modification of various plants, such as Arabidopsis, rice, and tobacco. With the establishment of tobacco gene editing breeding factories and the rapid promotion of material creation, massive gene editing materials are created, and the editing materials become important points in tobacco plant phenotype screening, but the tobacco leaves with similar tobacco plant phenotypes have differences in chemical components and physiological and biochemical indexes. Therefore, how to carry out efficient and accurate screening on tobacco plants on massive editing materials becomes a problem to be solved urgently.
The screening of tobacco plants relates to multiple steps of tobacco plant selection, tobacco sample collection, processing analysis feedback and the like, and comprises tobacco plant selection requiring tobacco sample collection, tobacco plant sampling marking, special phenotype recording, tobacco collection bagging, tobacco cryopreservation, tobacco freeze drying, tobacco grinding, tobacco powder storage, detection and residual sample treatment, and finally re-screening of massive tobacco plants according to feedback of detection results of tobacco. Meanwhile, the sampling and pretreatment processes have great randomness, and instability can be brought to subsequent detection if unified management is not performed. These seriously affect the establishment of tobacco gene editing and breeding factory and the rapid promotion of material creation.
The present invention has been made to solve the above problems.
Disclosure of Invention
The invention discloses a screening method of mixed library gene editing T2 generation tobacco plants, which can efficiently, accurately and conveniently manage the screening of tobacco plants collected by tobacco leaf samples, the collection, sign-in, processing, storage, dispatching and treatment of remaining test samples, thereby completing the efficient and accurate screening of massive tobacco plants of mixed library gene editing T2 generation. The technical scheme of the invention is as follows:
the invention discloses a screening method of mixed library gene editing T2 substitute tobacco plants, which comprises the following steps:
(1) collecting sample information by using a palm computer and wirelessly connecting the sample information with a server terminal;
(2) managing tobacco leaf samples according to the result of wireless connection between the palm computer and the server terminal, and completing operations of screening tobacco plants needing tobacco leaf sample collection, signing, processing, storage, sample sending, detection, remaining sample storage and remaining sample treatment in real time; and feeding the detection result back to tobacco plant growers, thereby completing the screening of mixed library gene editing T2 substitute tobacco plants.
Preferably, the step (2) comprises:
(21) dibbling and coding T1 generation seeds of the tobacco plant subjected to gene editing, performing DNA sequencing in the seedling stage, and uploading the DNA sequencing result of the tobacco plant and the corresponding tobacco plant code to a server terminal; the tobacco plant T1 generation seeds are seedling tobacco plants planted by the tobacco plant T0 generation seeds, and the tobacco plant T0 generation seeds are seeds of gene editing regeneration seedling tobacco plants;
(22) observing which tobacco plants reach the bud stage, scanning the tobacco plant codes in the bud stage by using a palm computer, and screening the tobacco plants according to the DNA sequencing result corresponding to the tobacco plant codes and whether the first collection is carried out or not to collect tobacco leaves so as to complete the screening operation of the tobacco plants;
(23) collecting tobacco leaf samples, filling the tobacco leaf samples into sample bags, sealing the sample bags, connecting a portable printer through Bluetooth to carry out sample coding and printing, attaching the printed sample codes to the sample bags, and uploading the sample codes to a server terminal, so that the operation of collecting the tobacco leaf samples is completed;
(24) the tobacco sample processing department processes the collected tobacco samples, then stores the tobacco samples in fixed positions corresponding to the codes, and adopts the palm computer to scan the sample codes and upload the stored positions to the server terminal, thereby completing the tobacco sample processing and storing operation;
(25) the tobacco sample dispatching department dispatches the stored tobacco samples to corresponding detection personnel and uploads the tobacco samples to the server terminal, so that tobacco sample dispatching operation is completed;
(26) the rest samples are returned to a sample storage department after detection is finished, and the storage department uses a palm computer to carry out coding and then stores the coded samples and uploads the coded samples to a server terminal;
(27) and the sample detection personnel records the detection result on a computer and uploads the detection result to the server terminal, and the tobacco plant planting personnel completes the screening work of mixed library gene editing T2 substitute tobacco plants according to the detection result and the record condition of the phenotype of the tobacco plants.
Still preferably, said step (21) comprises:
(211) carrying out tagging coding on the differentiated seedlings after gene editing, wherein each differentiated seedling has a corresponding unique code;
(212) molecular sampling and DNA sequencing are carried out in the seedling stage, and the sequencing is carried out in two steps: the method comprises the steps of firstly sequencing whether the tobacco plant has sgRNA insertion or not, and secondly sequencing whether the tobacco plant has gene editing or not;
(213) and uploading the DNA sequencing result of the tobacco plant and the corresponding tobacco plant code to a server terminal.
Further preferably, said step (22) comprises:
(221) scanning the tobacco plant codes by using a palm computer, judging whether the tobacco plants are the first tobacco leaf collection according to a server terminal, and if so, judging that the tobacco plants need to collect tobacco leaf samples; otherwise, not collecting the tobacco leaf sample;
(222) and (4) obtaining the tobacco plants which need to be collected in the step (221), continuously scanning the tobacco plant codes by using the palm computer, and judging whether to collect tobacco leaf samples according to the DNA sequencing result corresponding to the server terminal.
Still further preferably, said step (222) comprises:
(2221) judging tobacco plants according to the DNA sequencing result corresponding to the server terminal, and classifying the tobacco plants into four types, namely a first type: sampling is not needed, and the DNA sequencing result is not edited; the second type: sampling is needed, and the DNA sequencing result is edited and is a heterozygote; in the third category: sampling is needed and attention is focused, and the DNA sequencing result is edited and homozygote; the fourth type: sampling is required, wherein the sampling is a female parent tobacco strain which is not subjected to gene editing treatment;
(2222) and scanning the second type, the third type and the fourth type of tobacco plants and uploading the tobacco plants to a server terminal, thereby completing the screening operation of the tobacco plants.
Still preferably, said step (23), further comprises:
(231) recording whether the tobacco plants have special phenotypes on the palm computer, wherein the special phenotypes comprise whitening or yellowing of leaf color, waxiness and leather of leaf surfaces, retraction of leaf margins, and multiple branches of main stems;
(232) and uploading the special phenotype and the tobacco plant code corresponding to the special phenotype to a server terminal.
Still preferably, said step (24) comprises:
(241) the tobacco sample processing department performs freeze drying and crushing treatment on the collected tobacco samples, puts the tobacco samples into a sample storage tube, scans sample codes on the tobacco sample bags at the same time, and prints the sample codes and attaches the sample codes to the sample storage tube through a Bluetooth connection portable printer;
(242) and then storing the sample storage tube at a fixed position on a sample rack corresponding to the code of the sample storage tube, and uploading the storage position to a server terminal by scanning the sample code by using a palm computer, thereby finishing the processing and storage operation of the tobacco leaf sample.
Still preferably, said step (25) comprises:
(251) the tobacco sample dispatching department dispatches the stored tobacco samples to corresponding detection personnel, and simultaneously records the dispatched personnel, the dispatched sample amount and the residual sample amount after dispatching on a palm computer and uploads the samples to a server terminal;
(252) the system automatically reminds detection personnel to preferentially detect tobacco leaf samples which need to be sampled and focus on.
Still preferably, said step (26) comprises:
(261) the sample storage department judges whether the stored remaining sample reaches a preset storage period, and if so, the remaining sample is destroyed to finish the process; otherwise, the palm computer is used for coding and then continuously storing and uploading the code to the server terminal.
Still preferably, said step (27) comprises:
(271) the tobacco sample inspector records the detection result and the corresponding tobacco sample tobacco plant code on a palm computer and uploads the result to the server terminal; the detection is spectral analysis detection, conventional chemical component analysis detection or gene editing target point preset metabolite detection;
(272) the tobacco plant grower judges the difference between the uploaded detection result and the phenotype condition of the tobacco plant and the existing tobacco plant which is not subjected to gene editing processing and is subjected to statistical analysis; if all the tobacco leaves are unchanged, the tobacco plants corresponding to the tobacco leaf samples are harvested and stored, are not further processed temporarily, and are recorded and uploaded to a server terminal; on the contrary, the tobacco plant corresponding to the tobacco leaf sample is harvested, the tobacco plants with the gene target spots preset with the metabolite detection result consistent with the DNA detection result are preferentially planted, the next round of tobacco plant screening is carried out, and the records and the uploading are carried out to the server terminal; thereby completing the screening work of mixed library gene editing T2 generation tobacco strains.
The invention has the beneficial effects that:
1. the method of the invention feeds back the screening of tobacco plants according to the detection indexes of tobacco leaf samples, so as to achieve the high-efficiency and accurate screening of similar massive tobacco leaf gene editing materials (namely tobacco plants). The method is not only suitable for screening of mixed library gene editing T2 substitute tobacco plants, but also suitable for screening management of mass tobacco plants at any stage. Meanwhile, the method can randomly determine the detection index and feed the detection index back to the tobacco plant grower, so that whether the tobacco plant corresponding to the tobacco sample is harvested and planted is screened out for the next round of tobacco plant screening.
2. In the method, tobacco plants are judged according to the DNA detection result in the tobacco leaf sample collection stage, the tobacco leaves are divided into four types, and the key attention category is provided, so that the sample collection workload is reduced, and the detection efficiency is improved.
3. In the method, the palm computer is adopted to scan the tobacco plant codes and automatically generate the sample codes in the tobacco sample collection stage, the field collected data is wirelessly connected with the information in the server terminal, the traditional process of manually recording the tobacco plant codes and inputting the tobacco plant codes into the computer is avoided, and the working efficiency is effectively improved.
4. According to the method, the position of the tobacco sample storage completely corresponds to the code, and the coding rule of the tobacco sample is fixed, so that the position of the tobacco sample storage is fixed, and the tobacco sample storage can be quickly inquired on a server terminal and a palm computer, so that shelf management can be performed on a large number of tobacco samples, and the accuracy of the tobacco sample storage position and the convenience of subsequent sampling are ensured.
5. According to the method, information is wirelessly connected with the server terminal and the information is communicated, so that the state of each tobacco leaf sample can be rapidly inquired at any time on the server terminal and the palm computer, the sampling, freeze drying, crushing and storing processes of the tobacco leaf samples can be systematically tracked, and the irretrievable performance during the sample circulation period is avoided.
Detailed Description
The technical scheme of the screening method of the mixed library gene editing T2 substitute tobacco plants comprises the following steps:
example 1
The invention provides a set of hardware devices used in field management in order to better achieve mixed library gene editing T2 substitute tobacco plant screening, and the hardware devices comprise a palm computer, a server terminal, a portable bar code printer and adhesive label paper.
The working process is as follows: the sample information collected by the palm computer is accessed to the database of the server terminal through the wireless network, and the data of the server terminal can be fed back to the palm computer through the original path at the first time to realize wireless connection and feedback, so that the sample information in the database of the server terminal is extended to the hands of each sample administrator. In the sample management process, the printing process of the label bar code is as follows: the palm computer drives the portable printer through the Bluetooth technology, so that the label bar code required by a printing position is printed.
The specific screening method of the mixed library gene editing T2 substitute tobacco plant comprises the following specific steps:
1. the palm computer information is wirelessly connected with the server terminal;
2. uploading the DNA sequencing result of the gene editing tobacco plant of the planted and grown tobacco leaves and the corresponding tobacco plant code to a server terminal by using a palm computer;
3. judging whether the tobacco plant is the first tobacco leaf collection and the second type of tobacco leaf to be sampled by using the palm computer according to the server terminal, wherein the DNA sequencing result is edited and is a heterozygote; then, collecting tobacco leaves;
4. the tobacco sample processing department freezes and crushes the collected tobacco samples, puts into the sample storage tube, scans the sample code on the tobacco sample bag at the same time, prints the sample code and pastes on the sample storage tube through the portable printer of bluetooth connection, then store the sample tube in the correspondent fixed position of code, and adopt the palm computer to scan the sample code and upload the storage position to the server terminal, thus finish the tobacco sample processing and storage operation;
5. the tobacco sample dispatching department dispatches the stored tobacco samples to corresponding detection personnel and uploads the tobacco samples to the server terminal, so that tobacco sample dispatching operation is completed;
6. and returning the residual samples to the sample storage department after the detection is finished, judging that the stored residual samples reach the preset storage period by the storage department, and destroying the residual samples to finish the process.
7. The tobacco sample inspector records chemical components detected by the corresponding tobacco sample tobacco plant codes on a palm computer and uploads the chemical components to a server terminal;
8. and (4) judging that the uploaded detected chemical components are the same as the chemical components coded by the existing tobacco plants which are not subjected to gene editing processing by the tobacco plant grower, temporarily reserving samples of the tobacco plants corresponding to the tobacco leaf samples for no further processing, and recording and uploading the samples on the palm computer to the server terminal.
Example 2
The hardware device is the same as the embodiment 1, and the specific screening method of the mixed library gene editing T2 substitute tobacco plants comprises the following specific steps:
a. the palm computer information is wirelessly connected with the server terminal;
b. uploading the DNA sequencing result of the gene editing tobacco plant of the planted and grown tobacco leaves and the corresponding tobacco plant code to a server terminal by using a palm computer;
c. judging whether the tobacco plant is the first tobacco leaf collection and the third type of tobacco leaf collection needing sampling and paying attention to the first time by using the palm computer according to the server terminal, wherein the DNA sequencing result is edited and is a homozygote; then, collecting tobacco leaves;
d. the tobacco sample processing department freezes and crushes the collected tobacco samples, puts into the sample storage tube, scans the sample code on the tobacco sample bag at the same time, prints the sample code and pastes on the sample storage tube through the portable printer of bluetooth connection, then store the sample tube in the correspondent fixed position of code, and adopt the palm computer to scan the sample code and upload the storage position to the server terminal, thus finish the tobacco sample processing and storage operation;
e. the tobacco sample dispatching department dispatches the stored tobacco samples to corresponding detection personnel and uploads the tobacco samples to the server terminal, so that tobacco sample dispatching operation is completed; the system automatically reminds detection personnel to preferentially detect tobacco leaf samples which need to be sampled and focus on;
f. and after the detection is finished, the residual samples are returned to the sample storage department, the storage department judges that the stored residual samples do not reach the preset storage period, and the residual samples are continuously stored and uploaded to the server terminal after being coded by using the palm computer.
g. The system automatically reminds detection personnel to preferentially detect tobacco leaf samples which need to be sampled and focus on; the tobacco sample inspector records physiological and biochemical indexes of the corresponding tobacco sample tobacco plant code detection on a palm computer and uploads the physiological and biochemical indexes to a server terminal;
h. the tobacco plant grower judges that the uploaded detected physiological and biochemical indexes are different from the physiological and biochemical indexes coded by the existing tobacco plants which are not subjected to gene editing processing, and then the tobacco plants corresponding to the tobacco leaf samples are harvested and planted for the next round of tobacco plant screening, recorded on a palm computer and uploaded to a server terminal; thereby completing the screening work of mixed library gene editing T2 generation tobacco strains.
Claims (9)
1. A screening method of mixed library gene editing T2 substitute tobacco strains is characterized by comprising the following steps:
(1) collecting sample information by using a palm computer and wirelessly connecting the sample information with a server terminal;
(2) managing tobacco leaf samples according to the result of wireless connection between the palm computer and the server terminal, and completing operations of screening tobacco plants needing tobacco leaf sample collection, signing, processing, storage, sample sending, detection, remaining sample storage and remaining sample treatment in real time; feeding the detection result back to tobacco plant growers, thereby completing the screening of mixed library gene editing T2 substitute tobacco plants; the step (2), which comprises:
(21) dibbling and coding T1 generation seeds of the tobacco plants subjected to gene editing, performing DNA sequencing in the seedling stage, and uploading the DNA sequencing results of the tobacco plants and the corresponding tobacco plant codes to a server terminal; the tobacco plant T1 generation seeds are seedling tobacco plants planted by the tobacco plant T0 generation seeds, and the tobacco plant T0 generation seeds are seeds of gene editing regeneration seedling tobacco plants;
(22) observing which tobacco plants reach the bud stage, scanning the tobacco plant codes in the bud stage by using a palm computer, and screening the tobacco plants according to the DNA sequencing result corresponding to the tobacco plant codes and whether the first collection is carried out or not to collect tobacco leaves so as to complete the screening operation of the tobacco plants;
(23) collecting tobacco leaf samples, filling the tobacco leaf samples into sample bags, sealing the sample bags, connecting a portable printer through Bluetooth to carry out sample coding and printing, attaching the printed sample codes to the sample bags, and uploading the sample codes to a server terminal, so that the operation of collecting the tobacco leaf samples is completed;
(24) the tobacco sample processing department processes the collected tobacco samples, then stores the tobacco samples in fixed positions corresponding to the codes, and adopts the palm computer to scan the sample codes and upload the stored positions to the server terminal, thereby completing the tobacco sample processing and storing operation;
(25) the tobacco sample dispatching department dispatches the stored tobacco samples to corresponding detection personnel and uploads the tobacco samples to the server terminal, so that tobacco sample dispatching operation is completed;
(26) the rest samples are returned to a sample storage department after detection is finished, and the storage department uses a palm computer to carry out coding and then stores the coded samples and uploads the coded samples to a server terminal;
(27) and the sample detection personnel records the detection result on a computer and uploads the detection result to the server terminal, and the tobacco plant planting personnel completes the screening work of mixed library gene editing T2 substitute tobacco plants according to the detection result and the record condition of the phenotype of the tobacco plants.
2. The method according to claim 1, characterized in that said step (21) comprises:
(211) carrying out tagging coding on the differentiated seedlings after gene editing, wherein each differentiated seedling has a corresponding unique code;
(212) molecular sampling and DNA sequencing are carried out in the seedling stage, and the sequencing is carried out in two steps: the method comprises the steps of firstly sequencing whether the tobacco plant has sgRNA insertion or not, and secondly sequencing whether the tobacco plant has gene editing or not;
(213) and uploading the DNA sequencing result of the tobacco plant and the corresponding tobacco plant code to a server terminal.
3. The method according to claim 1, characterized in that said step (22) comprises:
(221) scanning the tobacco plant codes by using a palm computer, judging whether the tobacco plants are the first tobacco leaf collection according to a server terminal, and if so, judging that the tobacco plants need to collect tobacco leaf samples; otherwise, not collecting the tobacco leaf sample;
(222) and (4) obtaining the tobacco plants which need to be collected in the step (221), continuously scanning the tobacco plant codes by using the palm computer, and judging whether to collect tobacco leaf samples according to the DNA sequencing result corresponding to the server terminal.
4. The method of claim 3, wherein said step (222) comprises:
(2221) judging tobacco plants according to the DNA sequencing result corresponding to the server terminal, and classifying the tobacco plants into four types, namely a first type: sampling is not needed, and the DNA sequencing result is not edited; the second type: sampling is needed, and the DNA sequencing result is edited and is a heterozygote; in the third category: sampling is needed and attention is focused, and the DNA sequencing result is edited and homozygote; the fourth type: sampling is required, wherein the sampling is a female parent tobacco strain which is not subjected to gene editing treatment;
(2222) and scanning the second type, the third type and the fourth type of tobacco plants and uploading the tobacco plants to a server terminal, thereby completing the screening operation of the tobacco plants.
5. The method according to claim 1, characterized in that said step (23) further comprises:
(231) recording whether the tobacco plants have special phenotypes on the palm computer, wherein the special phenotypes comprise whitening or yellowing of leaf color, waxiness and leather of leaf surfaces, retraction of leaf margins, and multiple branches of main stems;
(232) and uploading the special phenotype and the tobacco plant code corresponding to the special phenotype to a server terminal.
6. The method according to claim 1, characterized in that said step (24) comprises:
(241) the tobacco sample processing department performs freeze drying and crushing treatment on the collected tobacco samples, puts the tobacco samples into a sample storage tube, scans sample codes on the tobacco sample bags at the same time, and prints the sample codes and attaches the sample codes to the sample storage tube through a Bluetooth connection portable printer;
(242) and then storing the sample storage tube at a fixed position on a sample rack corresponding to the code of the sample storage tube, and uploading the storage position to a server terminal by scanning the sample code by using a palm computer, thereby finishing the processing and storage operation of the tobacco leaf sample.
7. The method according to claim 1, characterized in that said step (25) comprises:
(251) the tobacco sample dispatching department dispatches the stored tobacco samples to corresponding detection personnel, and simultaneously records the dispatched personnel, the dispatched sample amount and the residual sample amount after dispatching on a palm computer and uploads the samples to a server terminal;
(252) the system automatically reminds detection personnel to preferentially detect tobacco leaf samples which need to be sampled and focus on.
8. The method according to claim 1, characterized in that said step (26) comprises:
(261) the sample storage department judges whether the stored remaining sample reaches a preset storage period, and if so, the remaining sample is destroyed to finish the process; otherwise, the palm computer is used for coding and then continuously storing and uploading the code to the server terminal.
9. The method according to claim 1, characterized in that said step (27) comprises:
(271) the tobacco sample inspector records the detection result and the corresponding tobacco sample tobacco plant code on a palm computer and uploads the result to the server terminal; the detection is spectral analysis detection, conventional chemical component analysis detection or gene editing target point preset metabolite detection;
(272) the tobacco plant grower judges the difference between the uploaded detection result and the phenotype condition of the tobacco plant and the existing tobacco plant which is not subjected to gene editing processing and is subjected to statistical analysis; if all the tobacco leaves are unchanged, the tobacco plants corresponding to the tobacco leaf samples are harvested and stored, are not further processed temporarily, and are recorded and uploaded to a server terminal; on the contrary, the tobacco plant corresponding to the tobacco leaf sample is harvested, the tobacco plants with the gene target spots preset with the metabolite detection result consistent with the DNA detection result are preferentially planted, the next round of tobacco plant screening is carried out, and the records and the uploading are carried out to the server terminal; thereby completing the screening work of mixed library gene editing T2 generation tobacco strains.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103049725A (en) * | 2012-11-21 | 2013-04-17 | 安徽省农业科学院水稻研究所 | Crop variety identity card management method |
| CN104657809A (en) * | 2014-11-10 | 2015-05-27 | 广州纤维产品检测研究院 | Laboratory sample management method based on handheld wireless data collector |
| CN106222193A (en) * | 2016-07-26 | 2016-12-14 | 浙江大学 | A kind of recombinant vector and the screening technique without transgene gene editor plant |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103049725A (en) * | 2012-11-21 | 2013-04-17 | 安徽省农业科学院水稻研究所 | Crop variety identity card management method |
| CN104657809A (en) * | 2014-11-10 | 2015-05-27 | 广州纤维产品检测研究院 | Laboratory sample management method based on handheld wireless data collector |
| CN106222193A (en) * | 2016-07-26 | 2016-12-14 | 浙江大学 | A kind of recombinant vector and the screening technique without transgene gene editor plant |
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