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Small non-coding RNAs224 of Lactococcus lactis subsp. lactis Yf11

A Lactococcus lactis, RNAs224 technology, applied in DNA/RNA fragments, recombinant DNA technology, bacteria and other directions, can solve the problems of low activity and poor stability

Active Publication Date: 2019-07-09
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lactococcus lactis grows well in a pH-neutral environment, but as the fermentation time prolongs, the lactic acid metabolized by itself makes the pH of the fermentation broth decrease continuously, inhibiting the growth of bacteria, and the accumulation of lactic acid itself will also inhibit the synthesis of nisin ; while nisin is very stable at pH 2.0, with the increase of pH value, the worse the stability and the lower the activity

Method used

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  • Small non-coding RNAs224 of Lactococcus lactis subsp. lactis Yf11
  • Small non-coding RNAs224 of Lactococcus lactis subsp. lactis Yf11
  • Small non-coding RNAs224 of Lactococcus lactis subsp. lactis Yf11

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Experimental program
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Embodiment 1

[0027] Obtaining the Sequence of Non-coding Small RNA s224 (s224 for short) of Lactococcus lactis subsp. lactis YF11

[0028] 1. Library construction and sequencing

[0029] Small RNA library: After extracting the total RNA of the sample, use gel cutting to recover fragments (18-150nt), according to IlluminaTruSeq TM The SmallRNA Sample Preparation protocol was used to construct a small library, and the final library was sequenced by Illumina HiSeq2000.

[0030] Strand-specific library: remove rRNA from the total RNA of the extracted sample and break it into short fragments, synthesize two cDNA strands successively using mRNA as a template, modify the second cDNA strand appropriately, and build a sequencing library after PCR amplification Sequencing was performed with Illumina HiSeq2000.

[0031] 2. Data preprocessing and bioinformatics analysis

[0032] The raw data obtained by sequencing needs to remove impurity data: reads containing adapter, reads with a ratio of N gre...

Embodiment 2

[0061] The functional analysis of embodiment 2, s224

[0062] In order to analyze the role of s224 in the survival and tolerance of Lactococcus lactis, a s224 overexpression strain was constructed and related detection experiments were carried out.

[0063] 1. Construction and verification of s224 overexpression strain

[0064] Using the genome of Lactococcus lactis subspecies YF11 as a template, primers sRNA-F and sRNA-R were designed according to the nucleotide sequence of s224 for PCR1 amplification. The amplified product was purified by agarose gel electrophoresis, recovered with a DNA purification and recovery kit, and then digested with plasmid pLEB124 with BamHI and HindIII, and then ligated with T4 DNA ligase. The ligated product was transformed into TG1 Escherichia coli competent cells, and the transformed product was coated on an LB plate with erythromycin (150ug / ml) resistance. Use the primers General-sRNA-F and General-sRNA-R on the colonies on the plate for PCR2...

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Abstract

The invention discloses a small non-coding RNA s224 of Lactococcus lactis subsp.lactis YF11. The nucleotide sequence of the mall non-coding RNA s224 of Lactococcus lactis subsp.lactis YF11 is represented by SEQ ID NO.1 in a sequence table, or is a nucleotide sequence having a homology of 90% or more with the nucleotide sequence represented by SEQ ID NO.1 and related with acid resistance. An experiment result shows that after the over-expression of the small non-coding RNA s224 of Lactococcus lactis subsp.lactis YF11, s224 over-expressed Lactococcal lactis has obviously enhanced survival ability in acidic environment, has improved survival rate in an acidic fermentation liquid, and increases the stability of nisin in order to improve the output of the nisin.

Description

technical field [0001] The present invention relates to bacterial non-coding small RNA (small non-coding RNA, sRNA), in particular to a non-coding small RNA s224 of Lactococcus lactis subspecies YF11, a small non-coding RNA s224 containing Lactococcus lactis subspecies YF11 Carrier, Lactococcus lactis containing the carrier and application. Background technique [0002] Bacterial small non-coding RNAs (small non-coding RNAs) are between 50-500 nt in length. As important regulatory factors in bacteria, they are mainly encoded by the genome, but most of them do not encode protein products. sRNA plays a role in the response of cells to environmental stress, and plays a regulatory role by complementary pairing with mRNA molecules or interacting with corresponding protein molecules. At the same time, sRNA is regulated by global regulators, participates in many important bacterial physiological pathways, and is also an important node in many signaling pathways. Moreover, sRNA ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/74C12N1/21C12P21/02C12R1/46
CPCC07K14/315C12N15/113C12N2310/141
Inventor 财音青格乐乔建军周丹丹赵秀丽李艳妮朱宏吉吴昊
Owner TIANJIN UNIV